Mycotoxins can produce toxic effects on humans; hence, it is of high importance to determine their presence in food products. This work presents a reliable method for the quantification of 32 mycotoxins in cheese. The analysis procedure was optimized based on a QuEChERS extraction process and the ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) detection. The analysis method was validated for four cheese varieties (emmental, blue, brie and camembert) in terms of linearity, sensitivity, matrix effect, accuracy and precision. Satisfactory precision and accuracy values were achieved, with recoveries above 70% for most mycotoxins. The developed method was applied to the analysis of 38 commercial cheese samples. A high occurrence of beauvericin and enniatins were found, ranging from 31% for enniatin A to 100% for enniatin B. The ochratoxin A was detected in three samples at concentrations that may pose a risk to human health.