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Mycotoxins are toxic metabolites produced by various genera of fungi. Fungi belonging to species of the genera Aspergillus and Penicillium are the main source of ochratoxin A (OTA) in wine. The reduction of OTA in must and wine has been studied using physical, chemical, and biological technologies. In this paper, we report on the capacity of different cultures isolated from Tempranillo winegrapes contaminated with fungus to degrade OTA in tryptic soy broth (TSB) medium at pH 3.5 and 6.5. We studied the interaction between OTA and four isolates during alcoholic fermentation of red wine. This study showed that microorganism isolates reduced the content of OTA in TSB medium and in the wine experiment system. The bacterium Lactobacillus rhamnosus exhibited the greatest mean reduction of OTA in TSB medium at both pH values (55%). During the wine fermentation, the yeast Metschnikowia pulcherrima showed the most reduction of OTA (34%). Liquid chromatography with tandem mass spectrometry was used to identify OTA degradation products, particularly α-OTA (m/z = 257) and the sodium adduct of α-OTA with the loss of the coumarin group (m/z = 170). The presence of OTA in must and wines is a problem for food safety. The use of lactic acid bacteria and yeasts during the winemaking process presents an interesting practical solution.