The emergence of multidrug-resistant bacteria presents a significant global health risk, necessitating the exploration of alternative treatment options. This in vitro study aimed to evaluate the efficacy of Saudi honey Clover and Talh against Gram-negative and positive bacteria, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis and Methicillin-resistant Staphylococcus aureus. All tested bacteria were collected and two types of Saudi honey were diluted and stored. This is followed by microbiological identification, prolonged exposure to honey (10 times), antibiotic susceptibility testing, and biofilm formation assay. The findings offer valuable insights into potential antimicrobial properties of Saudi honey against bacteria. The results demonstrated that Talh and Clover Honey caused alterations in the antimicrobial sensitivity of the bacteria tested. In Gram-negative bacteria, E. coli treated with honey showed increased sensitivity to imipenem, while P. aeruginosa and K. pneumoniae became increasingly susceptible to cell wall inhibitors. In Gram-positive bacteria, adapted MRSA and S. epidermidis strains exhibited increased sensitivity to ampicillin, amoxicillin/clavulanic acid, and ceftriaxone. The results suggest that honey’s antimicrobial effects vary based on its components and bacterial species. Consequently, further analysis is required to uncover the underlying mechanisms of the observed changes. The complex interactions between honey and pathogens, as shown by varying bacterial responses and limited impact on biofilm formation, highlighting the necessity for alternative therapeutic approaches. This study contributes to the existing evidence on the antimicrobial properties of honey.