Zinc oxide (ZnO) has broad applications in different areas. Green synthesis is an alternative to conventional physical and chemical methods. In our research, zinc oxide nanoparticles (ZnO NPs) were synthesized by the use of leaf extract of strawberry plants under different parameters. ZnO NPs biosynthesis was confirmed with UV-visible spectrophotometer and then characterized by Scanning Electron Microscopy (SEM). The significant antibacterial and antifungal efficacy of ZnO NPs was attained. The synthesized ZnO NPs indicated antimicrobial activity against both Pseudomonas aeruginosa and Botrytis cinerea pathogens. Different concentrations of ZnO NPs were tested against P. aeruginosa. The results indicated that ZnO nanoparticles synthesized with strawberry leaves extract showed antibacterial activity against tested bacteria. The most effective concentrations were 26 and 42 mg/ml for non-calcinated and calcinated ZnO NPs, respectively. Different ZnO NPs concentrations caused inhibition in the fungal growth of B. cinerea, and the inhibition enhanced with the increase in the concentration of NPs. Overall, the results showed a rapid, cost-effective, environmentally friendly and convenient method for ZnO NPs synthesis, which could be used as a potential antimicrobial agent against bacterial and fungal diseases.