This study aimed to determine the effect of green bismuth oxide (BiO) NPs against multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) from wound infections. Among 450 wound samples collected from patients admitted to the hospital, 200 P. aeruginosa isolates were identified. MDR strains of P. aeruginosa were detected by disc diffusion method. BiO NPs were synthesized using wild Bacillus subtilis (B. subtilis) strain and infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques. The antibacterial effect of the NPs compared to antibiotics against MDR strains was evaluated using a standard disk diffusion method. BiO NPs were synthesized at 0.005 M concentration of solution. According to the SEM images, the BiO NPs were irregular in size and ranged from 23-41 nm. According to the disk diffusion method, the highest inhibitory effect of BiO NPs was observed against MDR P. aeruginosa at 2000 ppm. The MIC and MBC of Bi-O NPs at 2000 ppm and >2000 ppm was observed against 97/200 (47.5%) of MDR P. aeruginosa. Eighty percent and 20% of P. aeruginosa produced strong and moderate-level biofilms, respectively. In the presence of sub-inhibitory concentration (1200 ppm) of Bi-ONPs, 52 (26%) isolates produced strong-level biofilms (p=0.001) and 22 (11%) produced moderate-level biofilm (p=0.029), while 53% of remaining isolates produced weak biofilms. The results exhibited a significant decrease in biofilm formation in the presence of Bi-O NPs. BiO NPs exerted an antibacterial effect that 2000 ppm and had a significant inhibitory effect against P. aeruginosa biofilms