Antibacterial activity of piperlongumine was evaluated against 18 clinically isolated strains, including identified strains belongs to Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, using the agar-well diffusion method. All the clinical strains showed concentration dependent susceptibility towards the constituent piperlongumine (25, 50, 100μg/100μL). It exhibited significant zone of inhibition against both the clinical and ATCC and MTCC strains. The antibacterial activity was more pronounced against Klebsiella pneumonia (24.00±0.12 mm) and Pseudomonas aeruginosa (20.00±0.12 mm) while it was moderate on Staphylococcus aureus (16.47±0.18 mm). The isolated constituent was found to show better activity profile, which indicates that the isolated constituent might be responsible for the antibacterial activity. The antibacterial activity was assessed comparatively with the standard drug Ciprofloxacin.