The presence of mycotoxins in cheese is a significant concern due to their potential health risks. Mycotoxins can contaminate cheese through two main routes: indirectly via contaminated animal feed, and/or directly, because of mold growth on dairy products. It has been reported that cheese may contain metabolites of aflatoxin B1 such as aflatoxin M1 (AFM1), aflatoxicol (AFL), and, its precursor, sterigmatocystin (STC). This study presents a reliable method for the simultaneous determination of AFM1, AFL, and STC in cheeses made from ovine, goat, or buffalo milk. The method was developed using single liquid extraction, clean-up by an immunoaffinity column (IAC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. The method was subjected to initial validation according to EU regulations, which outline the required performance parameters and criteria of analytical methods for official food control. The limits of quantification (LOQs) of the method for AFM1, AFL, and STC are 2.0 ng/kg, 5.0 ng/kg, and 1.0 ng/kg, respectively. The method was applied in a study for the assessment of mycotoxin transfer from milk to cheeses and also their growth.