ObjectivesThere is a significant apprehension in medicine and dentistry concerning the emergence of antibiotic-resistant pathogens, as it composes a significant threat to global health, particularly oral health. The growing concern that oral pathogens may develop resistance against standard preventive measures raises the need for alternative measures to prevent these pathogens’ growth without inducing microbial resistance. Therefore, this study aims to assess the antibacterial properties of eucalyptus oil (EO) against two main oral disease pathogens,Streptococcus mutans, andEnterococci faecalis.MethodsS. mutansandE. faecalisbiofilms were initiated using brain-heart infusion (BHI) broth supplemented with 2% sucrose with and without diluted EO. After 24 h of biofilm formation, total absorbance was measured via spectrophotometer; then, the biofilm was fixed, stained with crystal violet dye, and measured at 490 nm. An Independentt-test was used to compare the outcomes.ResultsDiluted EO revealed significant total absorbance reduction againstS. mutansandE. faecaliscompared to the control (p≤ 0.001). For the biofilm measurement,S. mutansandE. faecalisbiofilms were reduced by around 60- and 30-fold, respectively, compared to the group with no EO (p≤ 0.001).ConclusionBased on this study’s results, using EO as an organic compound could be considered an adjunctive tool in preventing the growth of oral pathogens causing dental caries and endodontic infection.