Zinc oxide nanoparticles (ZnO NPs) are a novel and alternative biomaterial for active biomedical applications among all metal and metallic oxide nanoparticles due to less toxicity and biocompatibility with human cells. In this study, we studied the growth curve of Serratia marcescens and Enterococcus faecalis to identify the mid-log phase of the bacterial growth to perform the exposure with ZnO NPs for investigating the antibacterial efficacy. The INT assay was used to determine the anti-bactericidal efficiency of ZnO NPs against S. marcescens and E. faecalis. The results showed that both the test bacteria attained the mid-log phase at the 5th hour. The determination of minimum inhibitory concentration (MIC) demonstrated a higher efficacy of ZnO NPs on the Gram-positive bacterium E. faecalis compared to the Gram-negative bacterium S. marcescens. The present study reports a higher susceptibility of Gram-positive bacterium over Gram-negative bacterium to the treatment of ZnO NPs.