The unregulated administration of currently available antimicrobial agents resulted in overspreading of resistant microbial phenotypes. In this study, Mucor racemosus was used for biosynthesis of zinc oxide nanoparticles (ZnO NPs) through fungi-based ecofriendly approach. The biosynthesized of ZnO NPs was initially considered based on analytical practices including UV–vis spectroscopy and transmission electron microscopy (TEM). Additionally, their cytotoxicity and anticancer activity were analyzed using suitable cell lines and their antioxidant effect was also assessed. Microbiologically, their inhibitory activity was comparatively evaluated against various methicillinresistant Staphylococcus aureus (MRSA) and methicillinsensitive Staphylococcus aureus (MSSA). Characterization of ZnO NPs displayed a distinct maximum absorption peak at 320 nm appeared in the UV–vis. Also, TEM revealed predominantly spherical ZnO NPs with particle size distribution ranging from 15 to 55 nm (mean size ≃ 40 nm). The normal cell line (Wi-38) illustrated the biosafety of ZnO NPs, where results showed IC(50) of 197.2 µg/mL. Furthermore, ZnO NPs exhibited promising suppressive activity on Hep-G2 cancerous cell with IC(50) of 51.4 µg/mL. Besides, ZnO NPs displayed antioxidant activity where IC(50) was 69.2 µg/mL. As well, the minimum inhibitory concentrations of ecofriendly ZnO NPs against the tested MRSA and MSSA isolates were ranged from 32 to 512 µg/mL. Also, their minimum bactericidal concentrations against the tested MSSA was in lower range, 32–1024 µg/mL, than the recorded range, 128–1024 µg/mL, against the MSSA. Also, the crystal violet (CV) assay showed an eradication potential of the biosynthesized ZnO NPs on MRSA and MSSA biofilm in a range of 23.24–73.96% and 6.63–74.1%, respectively. In conclusion, the ecofriendly synthesized ZnO NPs with antioxidant and anticancer activities demonstrated promising inhibitory effect on planktonic growth form of MRSA and MSSA clinical isolates with capability to eradicate their preformed biofilm. To achieve their full potential, future research needs to enhance the synthesis process to make ZnO NPs more uniform and scalable, as well as investigate their action mechanisms at the molecular level.