In recent years, the interest in the exploitation of fungal metabolites has grown considerably, given their application in numerous sectors involving human health. However, their identification and characterization by conventional analytical approaches is generally limited to single families of molecules per method of analysis. This constitutes a limiting factor of primary importance in the study of both the metabolic pattern of a single fungal sample and the discovery of its possible applications. In this work, a reverse‐phase high‐performance liquid chromatography coupled with mass spectrometry method for the profile determination of primary and secondary metabolites produced by the oyster‐mushroom Pleurotus ostreatus (Jacq.) P. Kumm., 1871, has been developed. By using a concomitant extraction in three different polarity‐decreasing solvents, namely methanol, ethanol, and acetonitrile, this method allowed the simultaneous analysis of all extracted metabolites belonging to the widest possible range of chemical families, giving an advantage for both qualitative and quantitative determination of known and unknown compounds. The method appears to be valuable and robust for the study of complex matrices like raw fungi extract such as those of Pleurotus ostreatus cultivated on different substrates and/or exposed to multiple stressors.