The Liberal Democratic Party, which has ruled Japan for almost all of the postwar era, selected hardline conservative Takaichi as its new head in a weekend vote.
But she must win approval in parliament to become premier later in October.
That had been seen as fairly straightforward given her coalition had by far the most seats, though it was short of a majority.
But with partner Komeito's exit and opposition parties seeking to unite behind an alternative candidate, her path has suddenly become less certain.
Following a meeting with Takaichi on Friday, Komeito leader Tetsuo Saito said the two parties' 26-year partnership had broken down over the LDP's failure to respond to a political funding scandal that has dogged the ruling group for two years.
He said Komeito would not support Takaichi in the parliament vote, expected to be held in the second half of October.
Takaichi called Komeito's decision "extremely regrettable" but said she would do whatever she could to win parliament's backing.
The political turmoil comes before a number of Japanese diplomatic engagements, with multilateral summits in Malaysia and South Korea, and US President Donald Trump widely expected to visit Japan at the end of October.
The yen strengthened as much as 0.5 per cent to 152.38 per dollar after the news, after falling to an eight-month low this week as investors fretted that Takaichi's big spending plans would weigh on the world's fourth-largest economy.
With Komeito gone, Takaichi might seek to broker alliances with other parties such as the centre-right Innovation Party.
Meanwhile, the main opposition Constitutional Democratic Party has suggested it may support the charismatic leader from another opposition party Yuichiro Tamaki as a candidate to challenge Takaichi for the premiership.
Tamaki, head of the populist Democratic Party for the People, said in a post on X that he was "prepared to serve as prime minister" and was deeply honoured by the CDP's support.
Opposition parties can put forward their own candidates when parliament meets to vote on the next premier.
Any candidate who secures a simple majority in the first round wins approval, and if not, the two candidates with the most votes go into a run-off.
Together the CDP and DPP control 175 seats in the lower house, while LDP is the largest party with 196 seats, 37 short of a majority.
Takaichi's selection as LDP leader last week dampened market expectations for a near-term interest rate hike.
She is known for her staunch support of former Prime Minister Shinzo Abe's "Abenomics" stimulus policies.
Komeito secretary-general Makoto Nishida told a media conference that Japan had entered an "era of the multi-party system" and that his party would strive to become "the axis of centrist, reform-oriented political forces",
Komeito, a socially liberal party affiliated with a lay Buddhist organisation, had also criticised Takaichi's tough language on foreigners and visits to the Yasakuni Shrine - seen by neighbours as a symbol of Japan's past militarism.
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