They included the last batch of Japanese passengers to leave the ship while some foreign passengers were still waiting on board for chartered aircraft to be sent by their governments. Around 970 passengers disembarked earlier this week.
Television footage showed a driver in a white protective suit at the wheel of a bus with the curtains drawn so that passengers could not be identified.
They will be quarantined for two weeks near Tokyo, officials said.
With the disembarkation, a 14-day quarantine is expected to start for more than 1,000 crew still on board as many of them did not undergo quarantine because they were needed to keep the ship running.
They were preparing food and delivering meals to cabins, leading some critics to charge they were inadvertently spreading the virus throughout the ship, which has seen more than 600 cases of the potentially deadly Covid-19 outbreak.
Health minister Katsunobu Kato on Saturday defended Japan's on-board quarantine, telling a TV programme there was no medical facility large enough to admit more than 3,000 people at once.
At a news conference late Saturday, Kato said six Australian passengers tested positive after leaving Japan.
One of them had left for Australia without confirming Japanese test results, while two others were in close contact with infected people.
"But the remaining three were not in close contact with infected people and tested negative in Japan," Kato said, adding that Japanese health authorities would cooperate with their Australian counterparts to discover how the three were infected.
Kato also confirmed that a Japanese hospital on Saturday used the anti-influenza medication Avigan, also known as Favipiravir, to treat a patient infected with the virus.
Earlier in the day, Kato said the government would push for the use of such medicines if they were confirmed to be effective.
At the news conference, Kato apologised that 23 passengers disembarked from the ship with hundreds of others without properly being tested.
The 23 tested negative but were tested before an isolation policy began on February 5, Kato said.
"We deeply regret that our operational mistake caused the situation," Kato said, adding that the passengers would be tested again.
Outside the Diamond Princess, Japan has seen 105 cases of the new coronavirus. - AFP
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