Bismuth nanoparticles were synthesized using laser ablation technique by focusing solid bismuth target in distilled deionized water by 1064 nm and 532 nm laser radiations generated by Q-switched pulsed Nd:YAG laser, respectively. Synthesized nanoparticles were characterized using UV–Vis spectrophotometer, X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Atomic Force Microscopy (AFM). The XRD pattern of the synthesized sample was indexed as. UV–Vis spectrophotometer indicated that the peak of absorption spectra of bismuth nanoparticles located in UV-region (230 nm) and increased towards IR region with increase laser energy and laser wavelength. SEM and TEM exhibited spherical shape of bismuth nanoparticles with decrease in particles size with decreasing laser wavelength. The antibacterial activity was tested against Enterobacter and Proteus (gram-negative bacteria) and Streptococcus and Staphylococcus aureus (gram-positive bacteria). Synthesized bismuth nanoparticles exhibited inhibitory effect on both bacteria strains with best selectivity against Enterobacter and Proteus (gram-negative bacteria).