Aim To assess the antimicrobial activity of probiotics SK12 and SM18 on Streptococcus mutans and also to compare the antimicrobial activity of SK12 and SM18. Materials and methods Synthetic strains of Streptococcus mutans were used to study the antimicrobial activity of probiotics SK12 and SM18 using various tests such as disk diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). In disk diffusion, the zone of inhibition was measured to assess the antimicrobial activity. Chlorhexidine was used as a control for this test. The MIC and MBC were assessed at different dilutions of the probiotic sample (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL, 6.25 mg/mL, 3.12 mg/mL, 1.6 mg/mL, 0.8 mg/mL, 0.4 mg/mL, and 0.2 mg/mL). Result SM18 demonstrated 20 mm of zone of inhibition, whereas SK12 demonstrated 15 mm showing a less antibacterial activity in comparison to SM18. SM18 was found to be bactericidal and effective at a minimum concentration of 0.8 mg/mL, whereas SK12 was bactericidal and effective at a minimum concentration of 1.6 mg/mL. Conclusion Probiotics demonstrate antibacterial activity against cariogenic microflora. SM is 18 having a better antibacterial activity at lower concentrations than SK12 in reducing cariogenic microorganisms. Clinical significance: BLIS K12 and M18 both demonstrated an antibacterial effect on Streptococcus mutans , wherein the use of probiotic in caries prevention is found to be limited. Hence, it is suggestive to reap the bacterial effects of BLIS K12 and M18 in caries prevention. How to cite this article Chandrasekhar SN, Mallikarjun SB, Salim HP. Comparative Evaluation of Antibacterial Activity of Probiotics SK12 and SM18: An In Vitro Study. Int J Clin Pediatr Dent 2020;13(6):611–616.