Among several animals, Rattus rattus (rat) lives in polluted environments and feeds on organic waste/small invertebrates, suggesting the presence of inherent mechanisms to thwart infections. In this study, we isolated gut bacteria of rats for their antibacterial activities. Using antibacterial assays, the findings showed that the conditioned media from selected bacteria exhibited bactericidal activities against Gram-negative (Escherichia coli K1, Klebsiella pneumoniae, Pseudomonas aeruginosa, Serratia marcescens, and Salmonella enterica) and Gram-positive (Bacillus cereus, methicillin-resistant Staphylococcus aureus, and Streptococcus pyogenes) pathogenic bacteria. The conditioned media retained their antibacterial properties upon heat treatment at boiling temperature for 10 min. Using MTT assays, the conditioned media showed minimal cytotoxic effects against human keratinocyte cells. Active conditioned media were subjected to tandem mass spectrometry, and the results showed that conditioned media from Bacillus subtilis produced a large repertoire of surfactin and iturin A (lipopeptides) molecules. To our knowledge, this is the first report of isolation of lipopeptides from bacteria isolated from the rat gut. In short, these findings are important and provide a platform to develop effective antibacterial drugs.