With 621 positive cases, the Diamond Princess is easily the biggest cluster of infected people outside China, and Japan has faced mounting criticism for its quarantine arrangements as passengers disperse across the world.
The disease has now claimed more than 2,000 lives in China and spread panic worldwide.
Those newly diagnosed with the virus will be taken off the ship to hospital, and after being treated will have to undergo another quarantine.
Earlier, those who had tested negative for the virus began leaving the ship.
“I’m relieved… I want to take a good rest,” said a departing 77-year-old Japanese passenger, who declined to give his name. He said he would be boarding Japan’s famously crowded railway system to head home.
A fleet of yellow-dotted city buses, plus a dozen or so taxis, whisked away from the passengers, many of whom dragged their luggage behind them and waved to former ship-mates on balconies as they disembarked.
Latest figures from China showed the death toll surging beyond 2,000 with more than 74,000 infected, although the rate of new cases is slowing.
Hundreds of more cases have been reported in two dozen countries, including 20 in South Korea, a two-thirds rise, with a cluster of at least 16 around the southern city of Daegu.
Hong Kong also reported its second death from the virus, which has proved extremely infectious.
For the 500 passengers leaving the Diamond Princess after testing negative, a difficult 14-day quarantine period has come to an end after their dream cruise turned into a nightmare of fear and boredom, confined in many cases too small windowless cabins.
“Our last deep gratitude to the crews and captain for such amazing care… during the epic crisis… we can’t wait to see you again soon on board again,” tweeted passenger Yardley Wong, who left after 14 days cooped in a small cabin with her six-year-old son.
Many were left onboard with an anxious wait for test results that would allow them to disembark.
Asked how he felt seeing others disembark while remaining on the ship, American lawyer Matt Smith told AFP: “I need an emoji for envy.”
Michael Ryan, head of the World Health Organization’s emergencies programme, said the outbreak was “very serious” and could grow, but stressed that outside China’s Hubei, it was “affecting a very, very tiny, tiny proportion of people”.
But in Japan, some have raised concerns about allowing people from the cruise ship to board flights home or spread into the notoriously busy Japanese capital.
Kentaro Iwata, a professor at the infectious diseases division of Kobe University, blasted the on-ship quarantine as a “major failure, a mistake”.
“It is highly likely secondary infections occurred,” Iwata told AFP, saying scepticism from abroad of the quarantine was “the only natural”.
He later said in a video published online that he was self-quarantining after a brief visit to the ship, where he raised major concerns about the procedures on board.
“It was completely chaotic,” he said.