SummaryIncreasing emergence of drug‐resistant microorganisms poses a great concern to clinicians; thus, new active products are urgently required to treat a number of infectious disease cases. Different metallic and metalloid nanoparticles have so far been reported as possessing antimicrobial properties and proposed as a possible alternative therapy against resistant pathogenic microorganisms. In this study, selenium nanoparticles (SeNPs) synthesized by the environmental bacterial isolate Stenotrophomonas maltophilia SeITE02 were shown to exert a clear antimicrobial and antibiofilm activity against different pathogenic bacteria, either reference strains or clinical isolates. Antimicrobial and antibiofilm capacity seems to be strictly linked to the organic cap surrounding biogenic nanoparticles, although the actual role played by this coating layer in the biocidal action remains still undefined. Nevertheless, evidence has been gained that the progressive loss in protein and carbohydrate content of the organic cap determines a decrease in nanoparticle stability. This leads to an alteration of size and electrical properties of SeNPs along with a gradual attenuation of their antibacterial efficacy. Denaturation of the coating layer was proved even to have a negative effect on the antibiofilm activity of these nanoparticles. The pronounced antimicrobial efficacy of biogenic SeNPs compared to the denatured ones can – in first instance – be associated with their smaller dimensions. This study showed that the native organic coating layer of biogenic SeNPs functions in avoiding aggregation and maintaining electrostatic stability of the nanoparticles, thus allowing them to maintain efficient antimicrobial and antibiofilm capabilities.