Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.