Unintended infestation of food with viable mould irreversibly leads to spoilage of food. However, little data existed on the fatty acid composition and polar lipid content of mould grown on cheese and the potential influence of these parameters in mould-infested cheese. In this study, we thus focused on the fatty acids composition and polar lipid content of cheese, mould isolated from mould-infested cheese, as well as inoculation of cheese with cultures of isolated moulds. For this purpose, we stored three cheese samples (gouda, brown cheese, and parmesan) over extended periods in order to force mould infestation from air. The moulds grown on gouda and brown cheese were identified as Penicillium brevicompactum and Penicillium roqueforti, respectively, while a mixed culture was obtained from parmesan. From these contaminated samples, mould spores were isolated and cultivated. Finally, the (pure) cheeses were inoculated with mould spores. Three subsamples, (1) cheeses, (2) moulds isolated from cheese, and (3) cheeses infested with the isolated moulds were studied with regards to their fatty acid distribution in the neutral and the polar lipids. The different amounts of polar lipids of cheese (0.39–2%), moulds (11–30.7%), and mould-infested cheese (1.5–5%) allowed calculating the mould content of the mould-infested cheeses.