The goal of this study is to identify the optimum conditions for ochratoxin A (OTA) biodegradation by the supernatant of Agaricus campestris strain. The Plackett–Burman and Box–Behnken methods were used to determine optimum OTA degradation conditions of Agaricus campestris under various incubation conditions. The Plackett–Burman method was planned through 16 varied experiments with 15 variants. The three most potent variants, sucrose, yeast extract and wheat bran, were selected using the Box–Behnken methodology. Ochratoxin A biodegradation ratio of 46.67% has been specified in only 1 h under ideal growing conditions. This is the first report on the optimization of OTA biodegradation by Agaricus campestris. When compared to previously published articles, it can be asserted that Agaricus campestris has promise based on its OTA biodegradation ratio in only 1 h of reaction time.