Background: A successful endodontic treatment is aimed at the sterilization of the entire pulp space. The use of extracts from Rhamnus prinoides as a novel irrigating material for root canal has not been studied . Hence, the antimicrobial efficacy of the alcoholic extract of Rhamnus prinoides as an irrigation material against E. faecalis was evaluated in comparison with the 2.5% sodium hypochlorite (NaOCL) solution used for root canals of permanent teeth. Methods: A total of 30 single-rooted human permanent teeth were thoroughly cleaned, shaped, and disinfected. Then, each tooth was subjected to a two-week infection with Enterococcus faecalis at 37 °C . Afterward, the samples were divided into three groups (10 teeth per group): 0.9% normal saline, 2.5% sodium hypochlorite, and 250 µg/ml Rhamnus prinoides. Paper points were used before and after irrigation to collect samples. Bacterial growth was evaluated after 24 h. Bacterial colonies were counted. The data were examined statistically via one-way analysis of variance, Dunnett’s T3 post hoc test, and Shapiro–Wilk test. The level of significance was set at p<0.05.  Results: The 2.5% sodium hypochlorite and 250 µg/ml Rhamnus prinoides demonstrated higher effectiveness against biofilm of Enterococcus faecalis than normal saline (p<0.05). The percentage of antibacterial effectiveness were 94.094 ±3.342, 93.685 ±5.280, and 25.603 ±3.912. Conclusions: Rhamnus prinoides, when used as root canal irrigant solution, was effective the sodium hypochlorite against the E. faecalis biofilm.