Graphene oxide (GO) exhibits several characteristics worthy of biomedical applications. The current study aimed to synthesize graphene oxide nanoparticles (GONPs) for antibacterial and wound-healing efficacy evaluations. The GONPs were prepared using Hummer's method and characterized by FT-IR, UV-Vis, Raman, SEM spectroscopic, and electron micrograph-based analyses, respectively, to confirm the structure and morphology. The analyses revealed GONPs as flaky, nano-sheeted structures with irregular shapes and sizes that were stacked on top of each other. The clinical isolates of gram-negative, i.e., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, as well as gram-positive, i.e., Staphylococcus aureus, microbes were incubated for 24 h at 37 celcius with 62.5, 125, and 250 mu g/ml concentrations of the freshly prepared GONPs which showed significant antimicrobial activity. All the pathogens were comparatively more active to 250 mu g/ml concentration of GONPs with inhibition zone diameters reaching 17.06, 15.10, 31.00, and 18.36 mm, respectively, as compared with the 250 mu g/ml concentration of cephalexin. The S. aureus-infected mouse wound-infection model utilized for in vivo investigation showed healing, when treated, twice daily, for 15 days, with GONPs at 250 and 500 mg/kg doses. The wound's contraction was observed to assess the therapeutic efficacy of the GONPs. The average wound healing of the treated groups was higher than that of the control. The animal groups treated with 500 mg/kg GONPs performed the highest levels of healing, reaching 99% of the healed animals within 15 days. Current findings suggested the potential of GONPs to serve as an agent for wound healing with antibacterial activity.