Navalnaya's call from abroad for resistance to Putin comes less than a month before a presidential election that is almost certain to hand the Kremlin chief another six-year term.
In a nine-minute video message laced with rage, Navalnaya, 47, accused Putin of killing her husband and said he had cut away half of her heart in doing so and robbed their two children of a father.
"I want to live in a free Russia, I want to build a free Russia," Navalnaya said in the video message entitled "I will continue the work of Alexei Navalny".
"I urge you to stand next to me," she said. "I ask you to share the rage with me. Rage, anger, hatred towards those who dared to kill our future."
It was unclear where she was speaking from but she was not in Russia. Navalnaya attended a meeting of European Union foreign ministers in Brussels on Monday which was weighing imposing further sanctions on Russia over her husband's death.
Britain has also threatened unspecified consequences for Russia. By contrast, Donald Trump, who drew criticism as U.S. president for his praise of Putin, made his first public comment on the death of Navalny but cast no blame.
Navalnaya accused Russian authorities of hiding Navalny's corpse and of waiting for traces of the Novichok nerve agent to disappear from his body. She gave no evidence, but said her team would publish details of who killed her husband.
"Vladimir Putin killed my husband," Navalnaya said. "By killing Alexei, Putin killed half of me - half of my heart and half of my soul.
"But I still have the other half, and it tells me that I have no right to give up. I will continue the work of Alexei Navalny, continue to fight for our country."
Tests on Navalny's body will take 14 days to complete, Ivan Zhdanov, an ally, said on Monday, citing an investigator.
Navalny, 47, fell unconscious and died suddenly on Friday after a walk at the "Polar Wolf" penal colony above the Arctic Circle where he was serving a three-decade sentence, the prison service said.
The West and Navalny's supporters say Putin is responsible for Navalny's death. The Kremlin has denied involvement and said that Western claims that Putin was responsible were unacceptable.
Putin has made no public comment on Navalny's death but it has further deepened a gaping schism in relations between Moscow and the West caused by the nearly two-year Ukraine war.
Asked by reporters on Monday how Putin had reacted to news of the death, his spokesman Dmitry Peskov said: "I have nothing to add."
NEW OPPOSITION LEADER?
Navalny rose to prominence more than a decade ago by documenting and poking fun at what he said was the vast corruption and opulence of the "crooks and thieves" running Putin's Russia.
He was by far the most charismatic figure in Russia's disparate opposition - and he gained respect for returning to Russia after a 2020 poisoning in Siberia. Navalny said a Russian hit squad had smeared the Novichok nerve agent in his underpants, which the Kremlin denied.
Continuing Navalny's battle, though, is fraught with difficulty - and danger.
Any attempt to lead the opposition from within Russia is almost certain to lead to arrest, but any leader doing so from abroad would be cast by Moscow as a foreign puppet controlled by Western intelligence.
"If (Navalnaya) does this in Russia, she will have a high chance of ending up where her late husband ended up," Alexei Levinson, head of sociocultural research at Russian pollster Levada Center, told Reuters.
"If she does this from abroad, the effect will be the same as for all the rest of those who somehow try to influence the processes taking place in Russia from abroad, and in my opinion, do not achieve very much."
Russian authorities cast Navalny and his supporters as extremists with links to the CIA intelligence agency who were trying to destabilise Russia. They have outlawed his movement.
Navalnaya's video appeal was not reported by state media in Russia. An unidentified source was quoted by some Russian media as saying she could soon be declared a "foreign agent".
Navalnaya, a graduate of the prestigious Plekhanov Russian University of Economics, supported her husband in his battles with Russian authorities. She attended his many court appearances, stood beside him at rallies and waited for his release from various prison terms.
"I know it seems that it is no longer possible (to continue the struggle)," she said in the video clip. "But we need more. To gather all together in one strong fist and hit this crazed regime with it - Putin, his friends, bandits in uniform, thieves and murderers who crippled our country."
Nadezhda Tolokonnikova, founding member of the Pussy Riot feminist opposition group that rose to prominence about a decade ago with its anti-Putin performances, said the opposition must keep on fighting.
"A lot of people said that we lost our hope with the loss of Navalny ... But I believe that we got a new sense of responsibility," Tolokonnikova said.
"If Navalny was here, he would want us to continue fighting, and he would want us to never give up. So, I guess our role is to carry his torch and do whatever we can, every one of us, to bring the beautiful new Russia of the future into reality."
