Mugabe denounced her before party loyalists as leader of a "treacherous cabal" bent on removing him from power.
In a thunderous speech to 12,000 cadres of his ZANU-PF party, Mugabe threatened to turn the law on Vice President Joice Mujuru, whose status as presumed successor for Africa's oldest head of state has evaporated in the past three months since she became the target of attacks in state media.
"As we thought we were working together, they were doing their own thing, a cabal parallel to the party, planning their own future, planning how to change the leadership, planning about kicking the president out of power," Mugabe said.
His comments appear to end the future in the ruling party of Mujuru, seen by some in the Zimbabwean business community as a common-sense leader who could have helped restore relations with the West that fell apart during the latter half of Mugabe's 34 years in power.
Every time he mentioned 59-year-old's name, the crowd crammed into a cavernous tent in a dusty field near Harare's central business district erupted into jeers and cackles.
"Those who are or were involved in corrupt activities, you are going to be prosecuted wherever we have enough evidence," he continued in the Shona language, accusing Mujuru of "gross corruption" in several gold and diamond mining deals.
He provided no evidence.
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Mujuru, a former guerrilla with the nom de guerre "Spill Blood", was not present at the meeting, a ruling party congress that is meant to anoint its leadership for the next five years.
A source close to Mujuru said she was watching the speech on television at her Harare home and would not be commenting.
Her only response during the three-month campaign against her by state media and Mugabe's 49-year-old wife, Grace, has been a short written statement in which she has denied allegations of corruption and plotting an assassination attempt.
CROCODILE LIES IN WAIT
Despite his advanced years and rumours of cancer, Mugabe is running unchallenged as ZANU-PF leader. After his speech, Mugabe stood on the podium decked out in a bright yellow jacket and baseball cap, swaying gently from side-to-side and clapping to the rhythm of a deafening jazz band.
Assuming he is still fit and able, he will be ZANU-PF's leader when the next elections come round in 2018.
With Mujuru gone, the succession race is more open.
Despite her political inexperience, Grace Mugabe is being elevated to the head of the powerful ZANU-PF Women's League, giving her a seat on the party's Politburo, its top decision-making body.
However, the one-time government typist faces an uphill struggle if she aims to succeed her husband, having played no part in the armed struggle in the 1970s against white rule in the former Rhodesia that is seen as conferring legitimacy on senior ruling party figures.
Justice minister Emmerson Mnangagwa, a 64-year-old career Mugabe loyalist nicknamed "The Crocodile" is seen as a likely candidate to assume the mantle of future successor.