Graphene oxide (GO) with zinc oxide nanoparticles (ZnO NPs) was synthesized using sol-gel and spin-coating procedures. This study aims to assess the antibacterial properties of ZnO-GO nanocomposites produced by spin coating with varying GO concentrations. So, different concentrations of GO NPs (10%, 30%, and 50%) were added to ZnO to assess the antibacterial activity. Structural and morphological properties of ZnO-GO were characterized using X-ray diffraction, field-emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy, and atomic force microscopy. The crystalline size of the pure ZnO NPs decreases with the increase of GO concentration, and the average crystalline size is 32.875 nm. The FESEM images showed that all samples contained mostly spherical-shaped particles with a high tendency to agglomerate. Also, the antimicrobial activity of the synthesized NPs (ZnO-GO) was investigated against the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, and the fungus Candida albicans. The antibacterial properties of three compounds were highly efficient. Additionally, it was demonstrated that ZnO-GO has higher antibacterial activity than pure ZnO NPs.