Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus spp. and Penicillium spp. that causes a threat to food safety and human health. Fungal biodegradation might be a promising strategy for reducing the OTA contamination in the future. In this study, the ability of Trichoderma koningii strains to degrade OTA produced by Aspergillus niger T2 (MW513392.1) isolated from tomato seeds was investigated. Among T. koningii strains tested, three strains; AUMC11519, AUMC11520 and AUMC11521 completely eliminated OTA from the culture medium, while AUMC11522 strain eliminated only 41.82% of OTA. OTα-amide, 3-phenylpropionic acid, OTα and phenylalanine were assayed as degradation products by FTIR analysis and LC–MS/MS spectra. Carboxypeptidase A (CPA) was found responsible for OTA degradation when a metal ion chelator, EDTA, was added to cell free supernatants of the three efective strains. OTA detoxifcation by T. koningii could present new prospective strategies for a possible application in food commodities intoxicated with ochratoxin.