Earlier this week, a high court judge dismissed Locadia Tembo's claims to be Mr Tsvangirai's customary wife.
But now a magistrate has agreed with her and ordered the wedding, which several heads of state were due to attend, to be put on hold.
Mr Tsvangirai's lawyers are seeking an urgent order to proceed with the event.
Also on Friday, they won another case - against Nozipho Shilubane from South Africa, who claims Mr Tsvangirai had promised to marry her.
Some analysts say the court cases bear all the hallmarks of a political dirty tricks campaign to smear the reputation of Mr Tsvangirai ahead of next year's elections, in which he is due to challenge President Robert Mugabe.
But others say he only has himself to blame as he has been involved with a number of women since his wife died in a car crash soon after he became prime minister in 2009.
President Mugabe, who is in a fractious coalition with Mr Tsvangirai, is due to attend Saturday's wedding to Elizabeth Macheka, 35, the daughter of a local politician from Mr Mugabe's Zanu-PF party.
The BBC's Brian Hungwe in the capital, Harare, says the ceremony had been set to take place in an upmarket area north of Harare.
Mr Mugabe is due to host a post-wedding banquet for other heads of state who will be there, he says.
Mr Tsvangirai, 59, and Mr Mugabe are expected to run against each other in presidential elections, due next year.
Although the high court judge dismissed Ms Tembo's case, he did say it should have been dealt with by a marriage officer at a magistrates court.
Ms Tembo, 39, a commodity trader and sister of a Zanu-PF MP, had demanded $15,000 (£9,310) a month in maintenance expenses from the prime minister.
In November, Ms Tembo and Mr Tsvangirai reportedly held a traditional wedding, and Mr Tsvangirai had paid a bride price of several hundred thousand dollars.
Shortly thereafter, Mr Tsvangirai said their relationship had been "irretrievably damaged" after it was "hijacked" by his opponents, including state security agents.
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