In this study, zinc oxide nanoparticles (Zn-NPs) were prepared by the green synthesis method and loaded inside niosomes as a drug release system and their physicochemical and biological properties were determined. Zn-NPs were prepared by the eco-friendly green strategy, the structure, and morphological properties were studied and loaded into niosomes. Subsequently, different formulations of niosomes containing Zn-NPs were prepared and the optimal formulation was used for biological studies. Scanning electron microscope (SEM) and dynamic light scattering (DLS) were used to investigate the morphology and size of nanoparticles. Fourier transform infrared spectroscopy (FTIR) and UV–Vis were used to confirm the synthesis of Zn-NPs. Energy dispersive X-ray spectrometer (EDS) determined the elemental analysis of the Zn-NPs synthesis solution and the crystalline structure of Zn-NPs was analysed by XRD (X-Ray diffraction). Furthermore, Zn-NPs were loaded inside the niosomes, and their structural characteristics, entrapment efficiency (EE%), the release profile of Zn-NPs, and their stability also were assessed. Moreover, its antimicrobial properties against some microbial pathogens, its effect on the expression of biofilm genes, and its anticancer activity on the breast cancer cell lines were also determined. To study the cytocompatibility, exposure of niosomes against normal HEK-293 cells was carried out. In addition, the impact of niosomes on the expression of genes involved in the apoptosis (Bcl2, Casp3, Casp9, Bax) at the mRNA level was measured. Our findings revealed that the Zn-NPs have a round shape and an average size of 27.60 nm. Meanwhile, UV–Vis, FTIR, and XRD results confirmed the synthesis of Zn-NPs. Also, the EE% and the size of the optimized niosomal formulation were 31.26% and 256.6 ± 12 nm, respectively. The release profile showed that within 24 h, 26% of Zn-NPs were released from niosomes, while in the same period, 99% of free Zn-NPs were released, which indicates the slow release of Zn-NPs from niosomes. Antimicrobial effects exhibited that niosomes containing Zn-NPs had more significant antimicrobial and anti-biofilm effects than Zn-NPs alone, the antimicrobial and anti-biofilm effects increased 2 to 4 times. Cytotoxic effects indicated that when Zn-NPs are loaded into niosomes, the anticancer activity increases compared to Zn-NPs alone and has low cytotoxicity on cancer cells. Niosomes containing ZnNPs increased the apoptosis-related gene expression level and reduced the Bcl2 genes. In general, the results show that niosomes can increase the biological effects of free Zn-NPs and therefore can be a suitable carrier for targeted delivery of Zn-NPs.