Resistance to antibiotics is rapidly spreading across the globe, posing a new health-care challenge for all countries. Bacterial biofilms are three-dimensional formations made up of cells encased in a matrix of polymeric, making cells resistant to the drugs and the immune system. As a result, new tactics for inhibiting the production of the EPS matrix may lead to more efficient use of already available antibiotics. The mechanism of vitamins C effect on boosting the effectiveness of several anti-bacterial drugs was investigated in this study, the target isolates were obtained from University of Mosul/ Biology department/ bacterial culture collections and evaluated qualitatively and quantitatively. The isolates involved: Staphylococcus aureus, Escherichia coli ,Klebsiella sp., Serratia marcescens and Pseudomonas aeroginosa. Antibiotic sensitivity tests and biofilm producing assay results revealed that the majority of the isolates were resistant to a range of antibiotics and had a large capacity for biofilm formation when grown on a cover glass surface. Vitamin C is an antioxidant, a scavenger of active metabolites. The Minimum Inhibitory Concentration (MIC) of Vitamin C against selected isolates had been determined, and all further experiments used concentrations below the MIC. Our results showed that Vit.C pre-treatment enhance the bactericidal effect of antibiotic and increases bacterial susceptibility to antibiotics. Using light microscopy, experiments of sub inhibitory doses of Vitamin C revealed good suppression of selected isolates biofilm development on the cover glass surface. Vitamin C can be utilized as an antibiotic adjuvant in combination with antibiotic and has effective biofilm inhibition, caused by multidrug-resistant bacteria, according to evidence.