Nanoparticle (NP) green synthesis is gaining popularity and has been proposed as a potential substitute for chemical and physical processes. The current study reports the environmentally friendly, low-cost method of optimized zinc oxide nanoparticles (ZnO NPs) utilizing a crude metabolite from Corynebacterium sp. ATCC 6931. Aliquot pH 8, 35°C, and an 8:2 (v/v) ratio of bacterial supernatant to zinc nitrate solution were the best conditions for the formation of ZnO NPs. Transmission electron microscopy (TEM), UV-visible spectroscopy (UV-vis), X-ray diffraction (XRD), and Zeta potential analyses were used to characterize the biosynthesized NPs. The synthesized ZnO NPs had sizes ranging from 8 to 17 ± 1.23 nm were mainly spherical in form and had a positive charge of ≈ +18.9 mV. The biosynthesized ZnO NPs in the current study have been applied in antimicrobial applications. Using the agar well diffusion method, different concentrations of biosynthesized ZnO NPs (50 µg/mL, 100 µg/mL, and 150 µg/mL) were used to test the antibacterial activity of the characterized NPs against the Gram-positive and Gram-negative bacteria: Bacillus cereus, Corynebacterium sp., and Escherichia coli. Additionally, the minimum inhibitory concentration (MIC) test was applied, and the results showed that bacterial growth reduces as biosynthesized ZnO NPs concentration increases. Also, compared to Gram-positive bacteria, Gram-negative bacteria appeared to be more sensitive to ZnO NPs.