Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.