Ergosterol is the most important sterol produced by most fungi and it is used as a chemical indicator for the presence of colonising fungi. The aim of this study was to investigate the effect of temperature and seed moisture content on the deterioration of microbiological quality of rapeseed. In this study microbial contamination of rape seeds was assessed based on the determined ergosterol concentration. Experimental material comprised seeds of rape cv. Californium stored in a thermostat chamber (25±1 and 30±1°C) equipped with three hygrostats used to maintain constant relative humidity. Prior to the experiment, rape seeds were wetted to moisture contents of 10.5, 12.5 and 15.5%. Seeds were stored until their germination decreased below 75%. During storage, samples were collected for analysis at every 6 days. Analysis showed a considerable effect of seed moisture content and storage temperature on the increase in ergosterol concentration.