ObjectiveThis study aimed to evaluate the effects ofApis trigonaethanolic propolis and probiotic bacteriaLactobacillus acidophiluson the nucleic acid concentration in the extracellular polymeric substances (EPS) derived from biofilm of root canal bacteria.Materials and MethodsClinical bacteria of the root canal were cultured with ethanolic extract of propolis (EEP; 10 or 0.1%) andL. acidophilus. After the formation of biofilm was observed in the monolayer bacterial culture under several conditions, the enzymatic treatment and nucleic acid quantification were sequentially performed.Statistical AnalysisIndependentt-test and Mann–Whitney were performed following data normality to analyze the significant differences of the treatment effect on the nucleic acid concentration in EPS from the isolated biofilm.ResultsThe results showed that the nucleic acid concentration in EPS biofilm were not increased by coculture withL. acidophilusas probiotics. However, the treatment with 10% EEP could significantly increase nucleic acid concentration.ConclusionThis study suggested that the biosurfactants from probiotic bacteriaL. acidophilusmight be a promising candidate for endodontic treatment, arguably better than EEP in inhibiting biofilm maturation and complexity.