In the present study, the antibacterial and anti-biofilm activity of selenium nanoparticles (SeNPs) produced by Bacillus subtilis BSN313 was investigated. Agar diffusion and microdilution methods did not reveal significant antibacterial activity of SeNPs against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923 due to the larger size of SeNPs and higher electrophoretic mobility, zeta potential, and/or poor diffusion properties in agar solids. Therefore, the agar overlay method was introduced, and significant antibacterial activity was observed with this method at concentrations (SeNPs) of 200 µg/mL, 150 µg/mL, and 150 µg/mL against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923, respectively. Atomic force spectroscopy (AFM) results, elucidated the disintegrating effect of SeNPs against Gram-negative (P. aeruginosa ATCC 9027) and Gram-positive (S. aureus ATCC 25923) pathogens. The disintegrating effect of SeNPs was caused by the destruction of the bacterial cell (biofilm producing) by rupturing the cell membrane. The hydrophobicity and zeta potential of the planktonic and biofilm populations were measured. Higher values of hydrophobicity and zeta potential heterogeneity were found for the biofilm population than for the planktonic population. The SeNPs showed significant anti-biofilm activity against biofilm-producing pathogenic strains. The ability of SeNPs to disperse biofilms was greatest at a concentration of 700 µg/mL within 15 min against P. aeruginosa ATCC 9027 (85.7%), S. typhi ATCC 14028 (89.6%), and S. aureus ATCC 25923 (78.3%). While the positive control (ascorbic acid, 32 µg/mL) showed an average dispersion of 95.1% against the biofilms of the strains studied within 90 min. Based on these results, we propose SeNPs as suitable candidates for the treatment of biofilm-producing pathogens.