Staphylococcus aureus colonizes the skin and nasal orifices of both humans and animals. Biofilm-related infections and acquired antimicrobial resistance render this bacterium a major health concern. This study examined the biofilm-forming capacity of eight S. aureus isolates originating from humans, sheep, and food matrices in northern Algeria. The efficiency of sanitizers (povidone–iodine [PVI], peracetic acid [PAA], quaternary ammonium compounds [QACs] and chlorhexidine [CHX]) in inhibiting and detaching biofilms was also assessed. Biofilm-production ability was assessed using a tissue culture plate. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration were measured for each sanitizer, and antibiotic susceptibility profiles were determined by performing the Kirby-Bauer disk diffusion assay. All S. aureus isolates demonstrated the ability to produce biofilms, and among these, food and sheep mastitis isolates exhibited a strong capacity for biofilm formation. CHX exhibited the highest efficacy in bacterial biofilm inhibition and was considered more potent than the other sanitizers (P < 0.0001). Further, it exhibited lower MIC interval values (0.98-1.9 µg/g). However, MIC interval values for PVI, PAA, and QACs were 3125-25000, 39.06-156.25, and 0.98-3.9 mg/L, respectively. PVI did not affect biofilm detachment at different concentrations. All isolates were sensitive to methicillin (with mecA/mecC genes not detected), gentamicin, sulfamethoxazole–trimethoprim, vancomycin, and amikacin; however, two isolates were resistant to erythromycin and clindamycin. These findings demonstrated the biofilm-forming potential of S. aureus isolates and confirmed the effectiveness of sanitizers against S. aureus isolated from different sources.