Exeter City manager Matt Taylor says League Two clubs are facing their most critical coronavirus tests this week.
The play-offs start on Thursday, with return legs the following Monday, and the final on 29 June.
Players are being tested twice a week, and anyone with a positive result must self-isolate for seven days.
"That's the most important one because if you get a positive test then you're basically out of the whole play-off campaign," Taylor said.
"All the hard work that would have gone on previously would have been for nothing, so I'll be sat with my fingers crossed hoping we don't get any positive tests," he told BBC Radio Devon.
Exeter travel to Colchester United on Thursday before hosting the U's in the return leg, with the winners of the semi-final to meet Northampton Town or Cheltenham Town for a place in League One next season.
"The positive tests we've had so far, the players have had no symptoms, and that's important to mention as people worry about people's health in these situations," Taylor added.
"But we've been really pleased that there have been no symptoms that have come alongside the positive tests and no family members seem to be affected.
"That increases the fact that we can make the players comfortable and safe in our environment."
Travel on match day?
A 20-man Exeter playing squad will travel to Colchester by bus for the tie, while the coaching staff will follow in their own cars in order to observe social distancing rules.
As it stands, the players will make the 250-mile journey on the day of the game, although the club are trying to find a hotel that could accommodate the players the evening before.
"Travelling on the day will mean a really early start," explained Taylor.
"On a coach it's the best part of six hours, and to do that before such a big game is unheard of and would be totally different to our normal preparations.
"So despite a different environment and different circumstances, we've got to make it feel like a routine the players are used to, so if that hotel becomes available for Wednesday night the players will certainly stay there and the staff will drive up on the day."