Aims To investigate the effect of support and growth medium (GM) on Pleurotus ostreatus biofilm production, specific metabolic activity (SMA) and ultrastructure. Methods and Results Biofilms were developed on membranes covering a broad range of surface properties and, due to the applicative implications of mixed biofilms, on standard bacterial GM in stationary and shaken culture. Hydrophilic (glass fibre, Duran glass and hydroxyapatite) and mild hydrophobic (polyurethane, stainless steel, polycarbonate, nylon) supports were more adequate for biofilm attachment than the hydrophobic Teflon. Among the GM, sucroseasparagine (SA) was more conducive to biofilm production than LuriaBertani and M9. GM was more influential than support type on biofilm ultrastructure, and a high compactness was evident in biofilms developed on SA. Biofilms on Duran glass were more efficient than planktonic cultures in olive-mill wastewater treatment. Conclusions The main effects of support and GM variables and their binary interactions on both biofilm production and SMA were all highly significant (P<0 center dot 001): thus, the magnitude of the effect of each variable strongly depended on the level of the other one. Significance and Impact of the Study There is a lack of basic information regarding physiology and ultrastructure of P.ostreatus biofilms. To our knowledge, this is the first attempt to fill this gap, thus representing a basis for future studies.