Objective: The present study was aimed to investigate the preliminary phytochemical analysis and HPTLC profiling and the antibacterial activity of P. pterocarpum methanolic flower extracts against the bacteria isolated from human infections. Methods: The preliminary phytochemical screening was performed according to the Harborne method. HPTLC studies were carried out using Harborne and Wagner et al method. The methanolic flower extracts of P. pterocarpum were tested against Salmonella typhi (MTCC 733), Staphylococcus aureus (MTCC 96), Proteus mirabilis (MTCC 742), Bacillus subtilis (MTCC 441) and Escherichia coli (MTCC 443). The antimicrobial activity was tested through well diffusion method. Results: The phytochemical studies on methanolic flower extract of Peltophorum pterocarpum (DC.) Baker ex Heyne. revealed the presence of glycosides, flavonoids, phenolics, saponins, catechin and alkaloids. The HPTLC separation was achieved using ethyl acetate-methanol-ethanol-water (8.1: 1.1: 0.4: 0.8) as the mobile phase. The methanolic extract of P. pterocarpum showed four different Rf values 0.16, 0.31, 0.77 and 0.82 which indicated various glycosides present in the flower extract. The methanolic extract of P. pterocarpum showed the maximum zone of inhibition against Proteus mirabilis followed by Salmonella typhi. Conclusion: Bio-assay revealed the presence of specific and selective antimicrobial compounds in the fractions. Broad range activity of plant extracts as per observations in this study was due to presence of multiple antimicrobial compounds or synergic effects of these compounds. Therefore, standardization of active fractions and study for in vivo efficacy may result in development of better antimicrobial drugs.