Aim: To analyze the efficacy of three different concentrations of Ocimum sanctum extract against various microorganisms, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus acidophilus. Materials and methods: Ethanolic extract of Ocimum sanctum was prepared by the hot extraction method. The extract was diluted with an inert solvent, dimethyl sulfoxide to obtain 3 different concentrations (2.5, 5 and 10%) of the extract. 0.2% chlorhexidine was used as a positive control and dimethyl sulfoxide was used as a negative control. The extract, along with the controls, was then subjected to microbiological investigation to determine which concentration among the 3 different concentrations of extract gave a wider inhibition zone against S. mutans, S. mitis, S. sanguis, L. acidophilus. The zones of inhibition were measured in millimeters. Results: Ocimum sanctum leaf extract demonstrated maximum antimicrobial activity against microorganisms responsible for dental caries at the 10% concentration level although 5 and 2.5% were also effective. Maximum activity was seen against S. mutans and S. sanguis with 10% extract. Conclusion: Ocimum sanctum leaf extract was effective against all the microorganisms. How to cite this article: Pai RK, Bhat SS, Salman A, Chandra J. Use of an Extract of Indian Sacred Plant Ocimum Sanctum as an Anticariogenic Agent: An in vitro Study. Int J Clin Pediatr Dent 2015;8(2):99-101.