Bryophyllum pinnatum is a medicinal plant from the Crassulaceae family. It's traditionally used in Ayurvedic and folk medicine to cure various ailments, and its phytochemicals are used in the reduction of metal ions to produce nanoparticles. This study is aimed at the biosynthesis of nickel oxide nanoparticles using Bryophyllum pinnatum as a reducing, capping, and stabilizing agent, characterizing the synthesized nanoparticles, and studying the anti-microbial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa as well as fungi; Candida (...) albicans, and Aspergillus niger. The synthesized nanoparticle was confirmed by UV-VIS, FT-IR, SEM-EDX, and XRD. The UV-VIS indicated the presence of chromophore and a surface plasmon resonance occurring in the sample with a band gap of 4.38 eV. The FT-IR result revealed the stretching vibration of the Ni-O bonds at 715 cm-1. The SEM analysis showed the morphology of the nanoparticles, and the EDX revealed that NiO-NPs are composed of oxygen and nickel elements. The XRD confirmed a face-centered cubic structure with an average size of 16.377 nm. The antimicrobial analysis exhibited the tested minimum concentration against all the bacterial strains. The synthesized Nickel oxide nanoparticles are useful in inhibiting the growth of different types of bacteria. -/- . (shrink)