Bacterial biofilm has been considered a major cause of many pathogen outbreaks and antimicrobial resistance. Therefore, the current study aimed to investigate the prevalence of the most significant pathogens that can form biofilm in raw milk, estimation of antibiotic sensitivity, antibacterial and anti-biofilm activity of sodium bicarbonate (SB), besides, its effect on the expression level of the virulence genes involved in biofilm formation using real-time PCR, also its effect on organoleptic and chemical constituents of milk.  S. aureus and K. pneumoniae were isolated from 38% and 23% of the total samples, respectively, with a higher rate of biofilm production for K. pneumoniae than S. aureus which showed the more strong biofilm density. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SB were 125mg/ml and 500mg/ml for S. aureus, respectively while, 125 mg/ml and 250 mg/ml for K. pneumoniae. All isolates were sensitive to ciprofloxacin and resistant to β- lactams. SB showed antibacterial and anti-biofilm activity with higher efficacy against K. pneumoniae and down-regulated the expression level of biofilm-associated genes (icaA in S. aureus, markA, and fimAo gene in K. pneumoniae). SB enhanced the activity of β-lactam, sulfamethoxazole/trimethoprim, and tetracycline, but slightly suppressed the activity of ciprofloxacin and gentamycin. Furthermore, the addition of 0.3 % of NaHCO₃ is enough to preserve milk samples for up to 12 hours at room temperature, it was effective chemically and microbiologically by reducing the initial viable cell count, also neutralizing the acids produced by acid-producing bacteria. so, it can be used for short-term preservation of milk.