Glycinin, basic subunit and β-conglycinin were isolated from soybean protein isolate and tested for their antimicrobial action against pathogenic and spoilage bacteria as compared to penicillin. The three fractions exhibited antibacterial activities equivalent to or higher than penicillin in the next order; basic subunit>glycinin>β-conglycinin with MIC of 50, 100 and 1000 μg/mL, respectively. The IC(50%) values of the basic subunit, glycinin and β-conglycinin against Listeria\monocytogenes were 15, 16 and 695 μg/mL, against Bacillussubtilis were 17, 20, and 612 μg/mL, and against S. Enteritidis were 18, 21 and 526 μg/mL, respectively. Transmission electron microscopy images of L. monocytogenes and S. Enteritidis exhibited bigger sizes and separation of cell wall from cell membrane when treated with glycinin or basic subunit. Scanning electron microscopy of B. subtilis indicated signs of irregular wrinkled outer surface, fragmentation, adhesion and aggregation of damaged cells or cellular debris when treated with glycinin or the basic subunits but not with penicillin. All tested substances particularly the basic subunit showed increased concentration-dependent cell permeation assessed by crystal violet uptake. The antimicrobial action of glycinin and basic subunit was swifter than that of penicillin. The cell killing efficiency was in the following descending order; basic subunit>glycinin>penicillin>β-conglycinin and the susceptibility of the bacteria to the antimicrobial agents was in the next order: L. monocytogenes>B. Subtilis>S. Enteritidis. Adding glycinin and the basic subunit to pasteurized milk inoculated with the three bacteria; L. monocytogenes, B. Subtilis and S. Enteritidis (ca. 5 log CFU/mL) could inhibit their propagation after 16-20 days storage at 4 °C by 2.42-2.98, 4.25-4.77 and 2.57-3.01 log and by 3.22-3.78, 5.65-6.27 and 3.35-3.72 log CFU/mL, respectively.