Summary Comparative studies of functional properties among closely related mushroom species, supported by molecular identification, standard cultivation and extraction protocols, are not well documented. We compared antioxidant and antibacterial properties of standardised hydroalcoholic extracts of four Pleurotus species (P. levis, P. ostreatus, P. pulmonarius and P. tuber‐regium). Antioxidant properties were investigated using DPPH and ABTS radical scavenging capacity, total phenolic content, β‐carotene‐linoleic and ORAC assays. Antibacterial effect was assessed using the microplate method. The functional properties of standardised mushroom extracts were different in species studied. β‐carotene–linoleic acid and ORAC assays showed high antioxidant activity, particularly in P. ostreatus. Pleurotus tuber‐regium exhibited the lowest antioxidant activity in the ORAC assay (3316.0 μmol of trolox equivalent mL−1), but exerted the most potent bacteriostatic and bactericidal activity. Bacillus subtilis showed a high degree of susceptibility to a very low concentration (3.33 μg mL−1) of P. levis extract. Remarkable antioxidant and antibacterial properties were found in P. levis and P. tuber‐regium compared to the other species studied that are cultivated commercially. Standardised mushroom hydroalcoholic extracts from Pleurotus species, molecularly identified, showed valuable antioxidant and antimicrobial properties. Pleurotus levis represents a novel source of bioactive compounds, while P. tuber‐regium exerted the most potent bacteriostatic and bactericidal activity. Gram‐positive and Gram‐negative bacteria showed differing degrees of susceptibility to Pleurotus extracts.