This study aimed to assess and understand the protective effect of vitamin C against secondary caries formation around the margins of class V restorations with the help ofan in-vitro bacterial caries model. In this study, a total of 30 mandibular premolars were chosen and assigned to three groups (n=10). In this study, Class V composite restorations were prepared and along with pretreatment of the cavity walls with various solutions like Vitamin C (ascorbic acid solution), HEPES buffer solution and chlorhexidinedigluconate. After incubating specimens with Streptococcus mutans, dentin and enamel were assessed using Fluorescence Microscopy.The study showed Vitamin C (Ascorbic acid) inhibited the formation of secondary caries near the dentin resin interface. No inhibitory effects were observed in enamel margins. From the study, it could be concluded that the incorporation of ascorbic acid in the restorative procedure of resin composite may inhibit secondary caries formation. Neither the application of chlorhexidine nor HEPES buffer had much effect on the inhibitory effect on secondary caries.