Background. Aggregatibacter actinomycetemcomitans (Aa) plays a vital role in some destructive forms of periodontitis. While mechanical and chemical plaque control is the first step in periodontitis treatment, side effects of adjunctive chemical agents such as chlorhexidine (CHX) mouthwash have led to the application of natural alternatives with minimal side effects. Therefore, this study evaluated the antibacterial effect of the hydroalcoholic extract of Quercus infectoria (Qi) galls on Aa in vitro. Methods. The hydroalcoholic extract of Qi was obtained by the maceration method, and Aa bacterial strain was cultured. The inhibition zone diameter was measured through the agar well diffusion method. Also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined by the broth microdilution method. All the experiments were repeated three times. 0.2% CHX was used as a control. Results. The inhibition zone diameter of Aa increased with increasing concentration of Qi extract. While MIC and MBC values for CHX were 0.0039 and 0.0078 mg/mL, respectively, both MIC and MBC values of the Qi extract for this bacterium were similar, i.e., 2.5 mg/mL, which was significantly higherd. Conclusion. Since other in vivo studies have confirmed the other properties of this extract and its safety in terms of cytotoxicity and mutagenicity, hydroalcoholic extract of Qi may be used in mouthwashes or local delivery systems to affect periodontal biofilm.