"FIFPro totally disagrees with the decision of the Bulgarian club Lokomotiv Plovdiv to order their players and coaches to take lie detector tests," said the Dutch-based union in a statement, describing the tests as "dubious."
Bulgarian first division club Lokomotiv ordered the tests after a surprise 1-0 loss on Saturday to struggling Botev Vratsa, who had been bottom of the table after a run of five consecutive defeats.
Lokomotiv's owner Veselin Mareshki told local media on Tuesday he had to be sure there were no other factors behind the team's loss.
"If this tool were to be applied, then it should be used by the defence, not by the prosecutor," said FIFPro lawyer Wil van Megen.
"The result of this lie detector test should only be used as incidental evidence, not as the only piece of evidence.
"Many scientists have criticised the use of the lie detector. They are not convinced that this tool is the most accurate to determine whether someone is telling the truth or lying.
"Experts say that people can cheat at these tests, that people can be influenced while taking these tests, that these tests can give the wrong results. Simply put: lie detector tests are dubious."
FIFPro added: "Players are often the victims of match-fixing. Professional footballers could be involved, but match-fixing never starts with the players.
"Therefore, it takes much more than focusing on footballers to abolish match-fixing."