Activity of zinc oxide nanosheets (ZnO NS) synthesized using actinomycetes of Nocardiopsis sp. GRG1 (KT235640) was analyzed for anti-biofilm activity against multidrug resistant (MDR) strains of Proteus mirabilis BDUMS1 (KY617768) and Escherichia colt BDUMS3 (KY617770) uropathogens. Characterization of synthesized ZnO NSs was done using scanning electron microscopy (SEM), UV-vis absorption spectrum, fourier transform infrared (FTIR), X-ray diffraction (XRD) pattern, raman spectrum, and energy-dispersive X-ray spectroscopy (EDS). Biofilm formation was evaluated by tissue culture plate method (TCP) and congo red agar assay (CRA). It was observed that the synthesized ZnO NSs showed significant antibacterial activity against the selected MDR strains. Further, these synthesized ZnO NSs holds the capability to deliver antibiofilm activity against MDR strains such as P. mirabilis BDUMS1 (KY617768) and E. colt BDUMS3 (KY617770).