Antimicrobial resistance is a major emerging clinical and public health issue across the globe. Nanotechnology is an emerging eld and developing area of scienti c interest in the world. Earlier reports showed in vivo antibacterial activity of bilirubin. Therefore, in present study, bilirubin nanoparticles (BNP) were synthesized of ∼100 - 150 nm, spherical shape and negative charge. Antibacterial activity of BNPs at different concentrations (0.03, 0.1 and 0.3 %) was evaluated by agar well diffusion method against the gram-positive i.e. S. aureus and gram-negative i.e. E. coli bacteria. Tetracycline and enro oxacin used as positive control. Tetracycline and Enro oxacin at different concentrations exhibited zones of inhibition against both S. aureus and E. coli . However, BNPs as well as bulk bilirubin showed no in vitro antibacterial effect on S. aureus and E. coli , however, testing by broth method and at higher concentration as well as against other microorganisms can be explored in future.