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In this research, we developed undoped and aluminum-doped zinc oxide for antimicrobial and anticancer activities. This study focuses on the synthesis, characterization, and biological activities of zinc oxide nanoparticles (ZnO NPs) and aluminum-doped zinc oxide nanocomposites (Zn_1−xAl_xO NCs) at varying concentrations (x = 0, 0.25, 0.5, and 0.75 wt%) using the coprecipitation method. Various characterization techniques such as XRD, UV-Vis, FTIR, EDX, and SEM were performed to analyze the crystal structure, optical properties, functional group identification, elemental composition, and surface morphology. The antimicrobial activity test showed that Zn_0.75Al_0.25O NCs exhibited the strongest inhibition zone against <i>Bacillus cereus</i> compared to <i>Staphylococcus aureus</i> > <i>Pasteurella multocida</i> > <i>Escherichia coli</i>. Moreover, the cytotoxicity and cell viability of liver cancer (HepG-2), breast cancer (MCF-7), ovarian cancer (SKOV3), and normal liver cell lines) were evaluated using the MTT assay, demonstrating that Zn_0.75Al_0.25O NCs not only enhance cell destruction but also show low cytotoxicity and high biocompatibility at low concentrations. These results suggest that Zn_0.75Al_0.25O NCs could be a promising candidate for in vivo anticancer applications and should be further investigated.