Nowadays, the emergence of drug resistance is a major problem due to the limited bioavailability and unintended toxicity of antibiotics because of their non-specific targeting, which makes it difficult to eradicate pathogenic infections. Zinc oxide nanoparticles gain much attention as nanocarriers due to their non-toxic, eco-friendly, and economical cost to treat severe pathogenic infections. The purpose of this research was to produce and analyze zinc oxide nanoparticles (ZnONPs) functions using a green synthesis process to evaluate their anti-bacterial and biofilm inhibitory activities against gram-positive and gram-negative microorganisms. ZnONPs were formed via a green synthetic approach using a seed extract of soybean (Glycine max) and nanoparticle characterization was done using FESEM, X-ray diffraction, UV–VIS spectroscopy, and EDAX. The anti-bacterial and biofilm inhibitory activities of ZnONPs, levofloxacin, and levofloxacin-loaded ZnONPs were determined using an agar disc diffusion assay and a microtiter plate assay, respectively. Levofloaxcin-loaded ZnONPs showed more potent and statistically significant antibacterial activity than ZnONPs alone against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis. The bacterial inhibition zone of levofloxacin-loaded ZnONPs at 80 µg/ml concentrations was higher in comparison to the standard levofloxacin and ZnONPs alone, which showed levofloxacin-loaded ZnONPs possess effective antibacterial activity that prevents the growth of diverse microorganisms. Our findings revealed that levofloxacin-loaded ZnONPs have an effective range of antimicrobial effects and could be used for the eradication of pathogenic infections.