Pseudomonas aeruginosa is capable of producing biofilms on various surfaces, medical implants and burn wounds which inhibit the action of antimicrobial agents. This study was undertaken for the screening, identification and purification of a natural antimicrobial compound from the hospital waste sites against P. aeruginosa. The partial purification of the antimicrobial agent was performed with ethyl acetate from the culture supernatant of the isolate and further purified by HPLC. For the characterization of the active compound, we have used Fourier Transform Infrared Spectroscopy (FT-IR) and Electrospray Ionization mass spectrometry (ESI-MS) analysis. The purified compound was tested for anti-biofilm activity in vitro against P. aeruginosa and on various surfaces such as plastic, glass, and steel. The potent isolate was confirmed as Bacillus licheniformis strain RG1002. The HPLC purified compound was characterized as lichenysin using FTIR and ESI-MS analysis. This study identifies and characterize the potent antimicrobial agent against other important human pathogens such as Staphylococcus aureus, Salmonella typhi, Shigella flexneri and Escherichia coli. Purified lichenysin showed strong inhibitory effect against P. aeruginosa and other important pathogens and inhibits the biofilm formation of P. aeruginosa in vitro on various types of surfaces. The current study suggests the application of lichenysin as a protective coating on various indwelling medical devices and catheters to prevent the biofilm formation by P. aeruginosa.