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Abstract Background To assess the antibacterial activity of multi-strain probiotics supernatants (MSP); Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus as an intracanal medication on Enterococcus faecalis (E. faecalis) biofilm in a tooth model. Methods Sixty extracted human single-rooted teeth with single canals were instrumented, sterilized, and inoculated with E. faecalis. After 21 days of incubation, four specimens were randomly selected to validate the biofilm formation by scanning electron microscope (SEM). The remaining specimens were randomly divided (n = 14), according to the intracanal medication (ICM) received into: Ca(OH) 2 : calcium hydroxide paste (35% Ultra Cal XS Ca(OH)2), Probiotics supernatants: MSP in poloxamer gel vehicle Poloxamer: poloxamer gel vehicle and, Control: E. faecalis biofilm only. The tested groups were further subdivided into two equal subgroups (n = 7) according to the incubation period (24 h and 7 days). Shaved dentin chips were obtained and collected by H-files and paper points, respectively for bacterial culture. The antibacterial activity was assessed after each incubation period quantitatively and qualitatively using bacterial colony-forming units per milliliter (CFUs/ml) and SEM, respectively. Results The lowest CFUs/ml was found in Ca (OH)2 with a significant difference compared to other groups after 24 h. After 7 days, a similar outcome was found with a further significant reduction of CFUs/ml in all groups with no statistical difference between Ca(OH)2 and probiotics supernatants groups. Ca (OH)2 and Probiotics supernatants groups showed a significant (p

Abstract Background There is an increasing demand to find a potent antibacterial agent against endodontic pathogens with the least toxic effect. The study aimed to evaluate the antibacterial activity of Lactobacilli probiotics on Enterococcus faecalis (E. faecalis) in comparison to calcium hydroxide paste. Methods The study involved two stages; (stage one): determination of the antibacterial properties of three strains of Lactobacilli probiotics supernatants (PS); Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and a cocktail mix of the three supernatants against E. faecalis using agar well diffusion method in both solution and gel phase. The formed zones of inhibition (ZOI) were measured in millimeters (mm) and compared to each other. PS solution and gel of the largest ZOI were further compared with calcium hydroxide paste (35% Ultra Cal XS Ca(OH)2) This was followed by (stage two): assessment of the minimum inhibitory concentration (MIC) of the PS that showed the largest ZOI against E. faecalis by agar well diffusion assay for both PS solution and gel. Results All supernatants showed growth inhibition against E. faecalis, and the cocktail mix showed the largest ZOI. However, no significant difference was found between the supernatants in both the solution and gel phases (p > 0.05). Ca(OH)2 showed a significantly lower effect than both the cocktail mix solution and gel (p 0.05). Conclusion PS has an antibacterial effect on E. faecalis and was more effective than Ca(OH)2. Lactobacilli probiotics could be a promising antibacterial agent used as an irrigant or an intracanal medication.