Background Based on the increase in antibiotic-resistant pathogens, it is necessary to have various effective compounds, so as to prevent its proliferation of these pathogens. For this purpose, nano-materials such as bismuth oxide nanoparticles can be used. Objectives The aim of this study was to produce bismuth oxide nanoparticles by Bacillus licheniformis PTCC1320 and to determine the antimicrobial effects on methicillin-resistant Staphylococcus aureus species compared with some antibiotics. Materials and Methods In this study, 200 bacterial samples were collected from hospitalized patients with burn infections from the Burn Rescue Hospital, Tehran. Thereafter, 65 strains of methicillin-resistant Staphylococcus aureus were identified by their phenotype and genotype. A total of 92% of identified strains with the highest resistance to antibiotics were isolated. Bismuth oxide nanoparticles were synthesized by Bacillus licheniformis PTCC1320. FTIR spectroscopy, X-ray diffraction, and scanning electron microscopy (SEM) were used to analyze the extracellularly produced nanoparticles. Finally, the antibacterial properties of nanoparticles produced on the biofilm of some pathogens were examined. Results In the present study, cube-shaped bismuth oxide nanoparticles were formed in the size range of 29–62 nm. They were found to have antimicrobial activity on 16% of the isolated Staphylococcus aureus strains. The FTIR results showed the vibrational frequencies of bismuth oxide at 583, 680, 737, and 1630 nm. The XRD results also confirmed the structure of nanoparticles. Compared with antibiotics such as Ciprofloxacin, bismuth oxide nanoparticles had less affectivity on this resistant hospital pathogen. Increasing the concentration of bismuth oxide nanoparticles, increased its antimicrobial effect and decreased bacterial growth rate. Conclusion Compared with heavy metals, bismuth nanoparticles have very low antibacterial effects. Considering this feature, the use of less antibiotics can be achieved with bismuth nanoparticles in the treatment of infections, thereby reducing antibiotic resistance.