In the current study synthesized Cobalt oxide nanoparticles (Co3O4 NPs) using Lawsonia inermis plant extract as fuel. The synthesized Co3O4 NPs were characterized using UV–visible spectroscopy, DLS, FT-IR, XRD, FESEM, and EDX analysis. The synthesized Co3O4 NPs were confirmed by the XRD analysis and their average size was determined using DLS and FESEM analysis. The average size of the cobalt oxide nanoparticles obtained from FESEM analysis was found to be around 98 nm and strong signals of cobalt were captured in EDX images. The synthesized Co3O4 NPs were tested on bacterial and fungal strains of three concentrations viz 50, 100 and 170 ppm. An increase in the concentration of Co3O4 NPs shows potent antibacterial and antifungal activities. The higher concentration (170 ppm) showed good antimicrobial activity on all strains of bacteria and fungi than the lower concentrations (50 and 100 ppm) of Co3O4 NPs. The observed zone of inhibition values against all bacteria and fungi ranged from 0.3 to 3.0 mm and 20 ± 0.42 mm to 0.3 to 2.9 mm, respectively. Among them, Staphylococcus and Streptococcus aureus showed more zones of inhibition at 170 ppm concentration. Meyerozyma guilliermondii fungal strain showed more zone of inhibition at 170 ppm concentration.