Patulin (1) is a mycotoxin contaminant in fruit and vegetable products worldwide. Biocontrol agents, such as the yeast Rhodotorula kratochvilovae strain LS11, can reduce patulin (1) contamination in food. R. kratochvilovae LS11 converts patulin (1) into desoxypatulinic acid (DPA) (5), which is less cytotoxic than the mycotoxin (1) to in vitro human lymphocytes. In the present study, we report our investigations into the pathway of degradation of patulin (1) to DPA (5) by R. kratochvilovae. Isotopic labeling experiments revealed that 5 derives from patulin (1) through the hydrolysis of the γ-lactone ring and subsequent enzymatic modifications. The ability of patulin (1) and DPA (5) to cause genetic damage was also investigated by the cytokinesis-block micronucleus cytome assay on in vitro human lymphocytes. Patulin (1) was demonstrated to cause much higher chromosomal damage than DPA (5).