Multidrug resistant (MDR) S. aureus infections continue to account for excess mortality in hospital and community settings and constitute a rising global health problem. However, data on the efficacy and mechanism of actions of alternative solutions like silver nanoparticles in developing countries are lacking. This study investigated anti-staphylococcal activity of silver nanoparticles (AgNP) against local strains from Nigeria. A total 119 clinical isolates of S. aureus from five Nigerian laboratories categorized as MRSA (n = 52) and MSSA (n = 67) by PCR were studied. The MIC of AgNP produced by chemical reduction method and characterized by surface plasmon resonance absorbance and size equivalence by scanning electron microscopy was determined by microbroth dilution method. Its effect on protease activity and plasmids were also investigated. Baseline characteristics of the isolates revealed MDR phenotype of the isolates, carriage of diverse plasmids (15-32 kb) among the MDR MSSA, and mean extracellular protease activity of 24.8-55.7 U/mL. The chemically synthesized AgNP had a peak absorbance at 400 nm with a size equivalence of 4.58 nm. The MICs of AgNP against the isolates were 4.7 μg/mL and 4.9 μg/mL, respectively, for MRSA and MSSA (P > 0.05). The bactericidal effect of AgNP at 2.5-5 μg/mL on the MSSA and MRSA isolates was observed at 2.7-5.5 h post exposure in vitro. Further analysis revealed plasmid eviction in the MDR MSSA isolates exposed to 5 μg/mL AgNP and dose-dependent reduction in extracellular protease activity by 84.6-93.1%. Hemolysis of human erythrocytes by AgNP was not observed at the MIC range. Conclusion: This study revealed safety and efficacy of AgNP against clinical MDR S. aureus isolates from Nigeria, using plasmid eviction and protease inhibition as mechanisms of action.