Antimicrobial efficacy of nickel nanoparticles synthesized using leaf extract of Ocimum sanctum (NiGs) was investigated against pathogenic Gram-negative ( E. coli, K. pneumoniae, and S. typhi), Gram-positive ( B. subtilis, S. epidermidis) bacteria and fungi ( C. albicans, C. tropicalis, A. fumigatus, A. clavatus, and A. niger). 100 µg/mL NiGs showed maximum antimicrobial activity against tested pathogens compared to leaf extract and antibiotics. E. coli (25 mm) and C. albicans (23 mm) exhibited higher zone of inhibition at 100 µg/mL NiGs. MIC, MBC, and MFC values of NiGs against all tested pathogens ranged between 25 and 50 µg/mL. Growth of bacterial and fungal cells (10 5 cfu/mL) was completely inhibited at 50 µg/mL NiGs. E. coli and C. albicans have showed strong antimicrobial activity with 81% and 50% reactive oxygen species (ROS) production, 30 and 16 µg/mL protein leakage, and 95 and 82 U/L LDH leakages, respectively. Gram-negative bacteria and Candida species showed more sensitivity to NiGs at all concentrations tested (25–100 µg/mL) than Gram-positive bacteria and Aspergillus species, respectively. Microbial growth in the presence of NiGs and ascorbic acid confirmed the involvement of ROS in antimicrobial activity. Hence, NiGs induced ROS generation was attributed to the protein and LDH leakage from microbial membranes.