Background <p>Some dipeptides function as bioactive substances to prevent the illness such as hypertension and dementia. However, almost no dipeptides are known to directly contribute to prevention of bacterial infection.</p> Methods and Results <p>We examined the antibacterial activity of dipeptides to six species of Gram-negative bacteria and a Gram-positive bacterium by the colony-forming units measurement and found that serine-proline dipeptide acts as an antibacterial agent selective on <i>Enterobacteriaceae</i> bacteria. In the microscopic observation of purified peptidoglycan particles, serine-proline dipeptide selectively disrupted Gram-negative bacterium-type peptidoglycan particles. In the protein analysis, serine-proline dipeptide induced hypertrophy of the bacterial cell bodies of an <i>Enterobacteriaceae</i> bacterium <i>Klebsiella pneumoniae</i> and caused the leakage of proteins from the bacterial cells. On the contrary, proline-serine dipeptide had no influence on the survival of all bacterial species examined. Apart from this, serine-proline dipeptide turned out to elicit the antibacterial ability of an antibiotic polymyxin B to <i>Enterobacteriaceae</i> bacteria. Meanwhile, proline-serine dipeptide inhibited the antibacterial action of polymyxin B on <i>Enterobacteriaceae</i> bacteria. In the molecular dynamics calculation, serine-proline dipeptide molecules integrated around a polymyxin B molecule and retained the stretched conformation of polymyxin B molecule. Meanwhile, a proline-serine dipeptide molecule interacted with joint moiety between the ring and linear structures of polymyxin B molecule and retained the folded conformation of its molecule. In sum, active and inactive forms of polymyxin B on <i>Enterobacteriaceae</i> bacteria were suggested to take the stretched and folded conformations, respectively.</p> Conclusions <p>Serine-proline dipeptide may be beneficial to prevention of <i>Enterobacteriaceae</i> bacterial infection.</p>