Bismuth nanoparticles (Bi NPs) constitute a promising technology for combating infectious illnesses and bacterial resistance to antibacterial treatments. Bi NPs were synthesized by Pulsed Laser Ablation in Liquid (PLAL) using a 532 nm second-harmonic generation Nd:YAG laser. Bi NPs samples were prepared in 10, 35, 50, 75, and 90 ËšC water for various laser energies and number of laser pulses. Ultraviolet-visible (UV-Vis) spectra measurements revealed a characteristic plasmonic absorption band indicative of metallic bismuth nanosized particles. Field emission scanning electron microscopy (FESEM) analysis showed that the water temperature during the ablation process affected the size and morphology of Bi NPs. The average Bi NP size at 60 ËšC was 28 nm, considerably smaller than the 55 nm average observed at 10 ËšC. The antimicrobial properties of Bi NPs against two opportunistic pathogens, E. coli and S. aureus, were assessed. For S. aureus, the inhibition zone of Bi NPs prepared at 60 ËšC was greater than that of samples prepared at 10 ËšC. Further, Bi NPs exhibited lower potency against E. coli than S. aureus in both samples.