Graphene oxide (GO)-NPs possess excellent physicochemical and biological properties and could have prominent antimicrobial activities. We used solution-based Hummer’s method to synthesize pure, CA coated and PEG coated GO-NPs and the XRD revealed hexagonal lattice structure and the crystallite size of pure and coated GO-NPs calculated in the range of 7–16 nm. In addition, a spongy like surface in pure GO-NPs and randomly crumpled like surface by using the CA and PEG coating agents was also identified and SEM images irregular, non-uniform and rod shape morphology of the graphite powder. The GO in the UV region and the band gap decreased from 2.30 to 2.03 eV while different modes (Cdouble bondC and Cdouble bondO) on the surface of pure and coated GO-NPs were evident as indicated by Raman spectra. Finally, in-vitro antimicrobial activity was assessed against two-gram negative bacterial strains viz. A. hydrophilaand E. coli and one fungal stain F. avenaceum at 10 and 20 mg/mL concentrations. Our results indicated enhancement in the inhibition zones of pure GO-NPs from 8 to 17 mm, PEG coated GO-NPs from 8 to 20 mm and CA coated GO-NPs from 8 to 17 mm respectively. Overall the PEG coated GO-NPs had prominent antimicrobial activities and we recommend application of polymer coated GO-NPs for wound healing process.