Ochratoxin A (OTA) is a secondary metabolite produced by fungal pathogens such as Penicillium verrucosum, which develops in food commodities during storage such as cereals, grapes, and coffee. It represents public health concerns due to its genotoxicity, carcinogenicity, and teratogenicity. The objective of this study was to evaluate the ability of actinobacteria and their metabolites to degrade OTA and/or to decrease its production. Sixty strains of actinobacteria were tested for their ability to prevent OTA formation by in vitro dual culture assays or with cell free extracts (CFEs). In dual culture, 17 strains strongly inhibited fungal growth, although it was generally associated with an increase in OTA specific production. Seventeen strains inhibited OTA specific production up to 4% of the control. Eleven actinobacteria CFEs reduced OTA specific production up to 62% of the control, while no substantial growth inhibition was observed except for two strains up to 72% of the control. Thirty-three strains were able to degrade OTA almost completely in liquid medium whereas only five were able to decrease it on solid medium, and two of them reduced OTA to an undetectable amount. Our results suggest that OTA decrease could be related to different strategies of degradation/metabolization by actinobacteria, through enzyme activities and secretion of secondary metabolites interfering with the OTA biosynthetic pathway. CFEs appeared to be ineffective at degrading OTA, raising interesting questions about the detoxification mechanisms. Common degradation by-products (e.g., OTa or L-ß-phenylalanine) were searched by HPLC-MS/MS, however, none of them were found, which implies a different mechanism of detoxification and/or a subsequent degradation into unknown products.