| Summary: | Antimicrobial resistance is a major global health concern and one of the gravest challenges to humanity today. Antibiotic resistance has been acquired by certain bacterial strains. As a result, new antibacterial drugs are urgently required to combat resistant microorganisms. Species of <i>Trichoderma</i> are known to produce a wide range of enzymes and secondary metabolites that can be exploited for the synthesis of nanoparticles. In the present study, <i>Trichoderma asperellum</i> was isolated from rhizosphere soil and used for the biosynthesis of ZnO NPs. To examine the antibacterial activity of ZnO NPs against human pathogens, <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> were used. The obtained antibacterial results show that the biosynthesized ZnO NPs were efficient antibacterial agents against the pathogens <i>E. coli</i> and <i>S. aureus</i>, with an inhibition zone of 3–9 mm. The ZnO NPs were also effective in the prevention of <i>S. aureus</i> biofilm formation and adherence. The current work shows that the MIC dosages of ZnO NPs (25, 50, and 75 μg/mL) have effective antibacterial activity and antibiofilm action against <i>S. aureus</i>. As a result, ZnO NPs can be used as a part of combination therapy for drug-resistant <i>S. aureus</i> infections, where biofilm development is critical for disease progression.
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