Emergence of drug resistance in bacteria for an array of antibiotics has created a demand for the discovery of novel drugs. In this study, we mixed the existing antibiotic and nanoparticle to determine their combined effect on bacteria. The magnesium-doped zinc oxide nanoparticles (Mg-doped ZnO NPs) were prepared through co-precipitation method by cost-effective manner. X-ray diffraction patterns revealed that the synthesized Mg-doped ZnO NPs exhibited wurtzite hexagonal structure. The average crystallite size is 33 nm for Mg-doped ZnO NPs. The Zn–O stretching bands appeared at 416 cm−1 for Mg-doped ZnO NPs determined using FT-IR spectra. Mg-doped ZnO NPs form nanoflake-like structures with an average thickness of 62 nm that was confirmed in HRSEM. Elemental compositions of nanomaterials were identified by EDX spectra. In order to explore new strategies to develop the next generation of drug and antibiotics agents to control human pathogen. The enhancement of antibacterial activity was exhibited against the selected human bacterial pathogens S. aureus, E. lentum, P. vulgaris, and E. aerogens, when antibiotic (chloramphenicol) was mixed with ZnO:Mg NPs than the individual components. Hence, this study concluded the synergistic effect of antibiotic and nanoparticle, which could be used to develop viable antibacterial drug. The cytotoxic effect of Mg-doped ZnO NPs was examined in cultured (MCF-7) human breast cancer cells.