Ergosterol (ERG) is a major sterol constituent of most fungi. Its concentration is negligible in higher plants, but can be used as a chemical marker of the presence of fungal contaminations. In this study, ERG concentration was assessed in randomly collected samples of naturally contaminated grain (wheat, barley and oat) and in samples of grain (wheat, barley, triticale and oat) harvested after inoculation of heads with conidia of different Fusarium species. Wheat samples were analysed at three stages of grain development. The lowest ERG concentration was found in non-inoculated samples at the first stage of grain development. This concentration was increasing with grain ripening. In naturally contaminated samples collected after harvest, ERG concentration was lower in wheat than in barley and oat. ERG concentrations in inoculated samples varied significantly, but were always significantly higher than in naturally contaminated samples. In the above cereal samples it was much lower than the levels assayed in laboratory cultures inoculated with fungi from genus Fusarium. The content of ERG was also analyzed in milling products of small-grained cereals and other foodstuffs, where a considerable variation was observed. The lowest ERG amounts were assayed in flours with a high degree of purification, while the highest ones in case of flours and products with a low purification rate. The results indicate the potential application of HPLC combined with microwave-assisted extraction both when assaying samples with low ERG concentrations (naturally contaminated) and those characterized with high contents of fungal biomass (strongly infected, artificially inoculated). It also facilitates analyses of fungal biomass in technological processes, where results may be expected to vary considerably.