The biosynthesis of metal oxide nanoparticles has attracted attention because of their bioactivity, low cost, rapid reactivity, and simplicity. This study aims to synthesize and characterize the nickel oxide nanoparticles (NiO-NPs) by Punica granatum seed extract and evaluate their bioactivities and toxicity. The biosynthesized NiO-NPs physicochemical characteristics were explored using NTA, TGA, XRD, UV–Vis, and FT-IR, showing the formation of high-purity crystalline NPs with an average size of 10–60 nm. Furthermore, the antibacterial, antioxidant, and hemolytic activity of the biosynthesized NiO-NPs were explored. The results of this study showed that NiO-NPs possess a high antibacterial activity against Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive bacteria (methicillin-resistant Staphylococcus aureus and Aerococcus viridans). However, the NiO-NPs inhibitory effect on Gram-positive bacteria was higher than on Gram-negative bacteria. Moreover, NiO-NPs also showed a DPPH, ABTS, and H2O2 radical scavenging activity with non-hemolytic activity at low concentrations (< 250 mg/L) in a dose-dependent manner. Regarding the toxicity of NiO-NPs, an enhanced effect on seed development of Vigna radiate at low concentrations (5 mg/L) was observed, which can be utilized as an alternative to conventional agrochemicals. Also, the cytotoxicity analysis was performed in the L-929 normal fibroblast cell line using MTT assay and IC50 (125.5 mg/L), indicating that the biosynthesized NiO-NPs are biocompatible at different concentrations. The obtained data showed that the bioactivity of NiO-NPs might be due to their small size, which generates various active sites because of their high surface area. Generally, P. granatum seed extract showed ability as an efficient reducing and capping agent for NiO-NPs biosynthesis and played a vital role in enhancing their bioactivity.