Agaricus bisporus mushrooms are well known for their nutritional and medicinal values. A. bisporus is a source of protein (about 40% on a dry basis), ergosterol, several minerals, carbohydrate, and fat. The present study was conducted to investigate the effect of A. bisporus S-II extracts on human pathogenic bacteria in-vitro condition. Totally, three human pathogenic bacterial strains (MTCC culture type) were procured from the Institute of Microbial Technology, India. Out of these three bacterial strains, one was Gram-negative (namely P. aeruginosa MTCC741), and the other two were Gram-positive (B. cereus MTCC9786 and S. aureus MTCC740). Microdilution assay was applied for the evaluation of the minimum inhibitory concentration (MIC). The highest antimicrobial activity was observed in methanol extract (26.5%) against S. aureus MTCC740, compared to ethanol extract (17%). Similar results were obtained for P. aeruginosa MTCC741 (21.8%) and B. cereus MTCC9786 (15%) in methanol extract. Least microbial growth inhibition observed for B. cereus MTCC9786 (13.82%) followed by P. aeruginosa MTCC741 (14%), compared to control in ethanol extract. The highest antimicrobial activity up to 17% with ethanolic extracts recorded against S. aureus MTCC740. The MIC results in microtitre plates showed the growth inhibition of P. aeruginosa MTCC741 and S. aureus MTCC740 at extract concentrations of 15 mg/ml and 20 mg/ml, respectively. However, no MIC detected for B. cereus MTCC9786 below 20 mg/ml extract concentration. Regarding minimum bactericidal concentration, the bactericidal value for P. aeruginosa MTCC741 and S. aureus MTCC740 was obtained at 10 mg/ml concentration. The present study indicated that the extracts of the A. bisporus S-II mushrooms had promising antimicrobial activities against the tested organisms.