The zinc oxide nanomaterials (ZnO-NMs), owing to their broad biomedical applications have lately attracted the incredible interest in the development of therapeutic agents against microbial infections. In this contribution, we have biosynthesized ZnO-NMs with a size of ˜ 40 nm from the Bougainvillea flower extracts. The FTIR and SEM-EDX mapping analysis confirmed the size, shape and biogenic origin of ZnO-NPs. Furthermore, the purified ZnO-NMs were applied for antibacterial studies against susceptible and resistant bacterial strains and to elucidate the possible mechanism of their activity. The XTT assay and confocal imaging confirmed the ZnO-NMs materials anti-biofilm activities against medically important pathogens, i.e., S. aureus and E. coli. Moreover, the absence of cytotoxicity against healthy kidney cells (HEK-293) and erythrocytes confirmed their biocompatible nature. Furthermore, the biosynthesized ZnO-NMs showed potent anticancer activity against the breast cancer cell line (MCF-7). These biosynthesized ZnO-NMs are having excellent antimicrobial and anticancer activities and are highly biocompatible due to biogenic nature. During antimicrobial study, Zno-NMs showed excellent minimum inhibitory concentration 16 μg concentration againt E. coli, P. aeruginosa and S. aureus. While in anticancer activity, of ZnO-NMs with 15 μg/ml dose showed good response against MCF-7 cell line. Further, this killing was mechanically confirmed by ROS generation by the ZnO-NMs, which cause cell lysis by the peroxidation of membrane lipid. So, this biogenic ZnO-NMs can be used in the future for nanomaterial-based drug development.