The rampant increase in antibiotic resistance has created a global barrier to the treatment of multidrug-resistant infections. Biogenic synthesis of nanomaterials is a novel approach to producing nanostructures with biological resources. Algae are known to be clean, nontoxic, cost-beneficial, and environmentally acceptable. Chlorella vulgaris is a popular microalga for its broad applications in food, supplements, pharmaceuticals, and cosmetics. In this study, we used Chlorella vulgaris biomass lyophilized powder as our green resource for the biosynthesis ZnONPs. Chlorella vulgaris culture was harvested at the end of the logarithmic phase, and the biomass was lyophilized. ZnONPs were synthesized using lyophilized biomass and 20 mM zinc acetate dihydrate at a temperature of 70 °C and continuous stirring in a water bath overnight. At the end of the reaction, UV-Vis absorption of colloidal suspension proved the synthesis of ZnONPs. The physicochemical characteristics of nanoparticles were analyzed using FTIR, DLS, TEM, and XRD. Based on FTIR spectra. The antibacterial activity of green synthesized nanostructures was evaluated against methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The synthesized ZnONPs have oxygen-containing groups on the surface that show the synthesized nanoparticles' stabilization. The Zeta potential was -27.4 mV, and the mean particle size was measured as 33.4 nanometers. Biogenic ZnONPs produced in this method have a notable size distribution and excellent surface energy, which can have vast applications like antimicrobial potential in pharmaceuticals as topical forms. Additionally, in order to evaluate the antimicrobial activity of ZnO nanoparticles, we used MRSA and VRE strains and the results showed the anti-MRSA activity at 400 and 625 μg mL-1, respectively. Thus, these biogenic ZnO nanoparticles revealed a substantial antibacterial effect against multidrug-resistant pathogens, associated with several serious systemic infections, and have the potential as an antimicrobial agent for further study.