The acting chief of Taiwan's representative office in Macau has returned home after refusing to sign a document recognising China's sovereignty over the self-ruled island, Taipei said on Tuesday.
"The future situation is not optimistic," Taiwan's Mainland Affairs Council said in a statement, calling the Macau authorities "disrespectful and unfriendly".
It said four other Taiwanese employees whose visas are still valid will remain in the semi-autonomous city to keep the office running.
It comes a week after seven employees of Taiwan's trade office in Hong Kong left the financial hub following their refusal to sign a similar "one China pledge" acknowledging Taiwan as part of China.
Macau and Hong Kong abide by authoritarian China's view that democratic, self-ruled Taiwan is part of its territory and must be seized one day, by force if necessary.
Both have closed their trade offices in Taipei and Beijing has sought to pile diplomatic and economic pressure on Taiwan.
The Taiwanese council also said there was a protest "by pro-China people outside our office in Macau to smear" the Taiwanese government.
"This shows that the authorities and civil sector in Macau have been disrespectful and unfriendly to our side," it said.
The Macau government has not responded to an AFP request for comment.
According to Taiwan's Liberty Times newspaper, the protesters accused Taiwan's President Tsai Ing-wen of damaging the "prosperity and stability" of Macau and Hong Kong in a demonstration a few days before Chen Chia-hung's departure on Sunday.
Beijing cut official contacts with Taipei and began a concerted pressure campaign after the 2016 election of Tsai, as she views Taiwan as a sovereign state and not part of Chinese territory.
Tsai's government is also a vocal supporter of democratic principles and has quietly helped open its doors to some Hong Kongers trying to escape Beijing's crackdown on dissent after huge democracy protests rocked the city in 2019.
Last month, Hong Kong suspended operations of its trade office in Taipei and accused Taiwan of "grossly interfering" in its affairs, causing "irretrievable damage" to relations.
Macau followed in shutting its office earlier this month, saying it was having trouble getting visas for staff.
Both Hong Kong and Macau are semi-autonomous Chinese cities where Beijing decides foreign policy and is ramping up direct control in both former colonies.