On Monday, the red, white and blue stripes of Russia were held up by fans during a first-round clash between Ukraine’s Kateryna Baindl and Russia’s Kamilla Rakhimova on day one.
Ukrainian fans reportedly called security and police to the stands.
A Russian flag was also unfurled at Rod Laver Arena during Daniil Medvedev’s clash with American Marcos Giron.
“I strongly condemn the public display of the Russian flag during the game of the Ukrainian tennis player Kateryna Baindl at the Australian Open today,” Ukraine’s ambassador to Australia and New Zealand Vasyl Myroshnychenko wrote on Twitter.
“I call on Tennis Australia to immediately enforce its ‘neutral flag’ policy.”
Tennis Australia in their statement said their initial policy was to allow fans to bring the flags as long as it did not create any “disruptions”.
Russian and Belarusian players were banned from Wimbledon last year but are able to compete as individual athletes without national affiliation at the Australian Open.
Their flags are not displayed beside their names in TV broadcasts, as is the case for other players, and their nation is not indicated on draw sheets.
Belarus is being used as a key staging ground for Russia’s war in Ukraine, which Moscow terms a “special operation”.
Former Australian Ambassador to Ukraine Doug Trappett, who served in the role from 2015 to 2016, also hit out at Australian Open organisers.
“Embarrassing Australian Open – and it’s only day one,” he tweeted.
Belarusian Aryna Sabalenka said she understood how the display of the flags of the two countries might upset Ukrainian players.
“I really thought that sport is nothing to do with politics but if everyone feels better this way, then it’s OK,” the fifth seed told reporters after her first-round win on Tuesday.
“If Tennis Australia made this decision to make them feel better, OK. They did it, what can I do? I can do nothing.”