Abstract The aim of this study was to assess the in vitro antimicrobial effects of chlorhexidine digluconate (CHX), polyhexamethylene biguanide (PHBM), and octenidine dihydrochloride (OCT) on cariogenic microorganisms by using their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). CHX, PHBM, and OCT were diluted in distilled water to the final test concentrations. Using the in-tube dilution method, Streptococcus mutans, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Actinomyces viscosus were cultivated on blood agar and Mueller–Hinton broth (MHB) at 37°C for 48 h. They were read using a spectrophotometer to detect MIC. To determine MBC, samples in the range of the turbidity threshold after 24 h were transferred onto blood agar and evaluated for growth after 24 h. Different MICs and MBCs were observed in all disinfectants against each microorganism. The lowest MIC and MBC against S. mutans (60 mg/L) were obtained from PHBM. The lowest values against L. rhamnosus (15 mg/L, 30 mg/L), A. viscosus (30 mg/L), and L. acidophilus (15 mg/L, 30 mg/L) were determined by OCT. PHBM and OCT have the potential to be replaced with CHX because they were effective against cariogenic microorganisms.