Background: Dental caries is a predominant microbial infectious disease of the tooth structure initiated and propagated by Streptococcus mutans and S. sanguinis. Systemic administration of antibiotics has many drawbacks. To overcome the limitations, herbal remedies have sprung up as an effective and viable option and has emerged as one of the important strategies to reduce pain. Aim: to evaluate the antimicrobial efficacy of Atropa belladonna, Silacea and Amoxicillin against S.mutans and S.sanguinis biofilm formed on tooth substrate in vitro. Methods: 9 Extracted human primary teeth sectioned below the cemento-enamel junction were placed in the tissue culture wells exposing the crown surface to S. mutans and S. sanguinis to form a biofilm. At the end of 21 days all groups were treated with Atropa belladonna, Silacea and Amoxicillin and were analyzed quantitatively. The significant difference between the two groups were tested using ANOVA. P<0.05 was set as significant. Results: There was a significant difference in the reduction of colonies and CFU/ml, Silicea had a significant effect in the reduction of S. sanguinis, while Atropa belladonna significantly decreased the colonization of S. mutans on the 21st day (p < 0.05). Conclusion: The antimicrobial efficacy of Silicea and Atropa belladonna were superior to amoxicillin in reducing S.mutans and S.sanguinis colonization on the tooth surface and could be used as an alternative to conventional antibiotics.