Nashville SC's game against the Chicago Fire in the MLS is Back Tournament has been postponed until a later, undisclosed date.
The fixture was originally set for July 8 at 10:30 p.m. ET.
The league also announced on Tuesday that the match between FC Toronto and D.C. United, which was originally scheduled for July 10, will now take place on July 12 due to Toronto's delayed arrival in Orlando.
"Since arriving in Orlando on July 3, five Nashville players have had confirmed positive tests for COVID-19. Two of the players received the confirmed positive results during the weekend and three players received confirmed positive results last night. In addition, four other players have received inconclusive test results, and require further testing," MLS said in a statement on Tuesday.
"In conjunction with the club, MLS will continue to evaluate Nashville SC's participation in the MLS is Back Tournament following the results of additional testing."
The postponement of Nashville and Chicago follows the withdrawal of FC Dallas from the tournament on Monday, along with news that MLS MVP Carlos Vela opted against traveling to the tournament with LAFC.
Vancouver Whitecaps forward Fredy Montero made the same decision later along with four of his teammates, Lucas Cavallini, Georges Mukumbilwa, Tosaint Ricketts and Andy Rose.
Cavallini said in a statement: "This was an extremely difficult decision for me. I would love to be out on the field with my teammates fighting with everything I have to play for this club and community in Orlando. Unfortunately COVID-19 has had a very big impact, taking away two beloved members of my family. I feel that it is best that I remain home to support my loved ones at this challenging time."
MLS commissioner Don Garber said: "We're very, very focused on ensuring that nobody is playing in this tournament that shouldn't be there.
"Anybody who had a special situation that required attention is something we were working in conjunction with our clubs to ensure we could address the issues. In the case of Carlos [Vela], we are disappointed he can't be there -- he's our reigning MVP -- but he has a wife that has an at-risk pregnancy, and he wanted to be there in support of her and we respect that and wish he and his wife great health with the birth of their child."