This in vitro study focused on the antimicrobial activity of methanolic and aqueous extracts of three organs (stems, roots, and leaves) ofPistacia lentiscusagainst nine bacterial species, two fungal, and one yeast strain. A comparative study of the yield, high-performance liquid chromatography (HPLC) composition, and polyphenol content of the different extracts was conducted. The obtained data showed that the yield of the methanolic extracts (between 13% and 33.3%) was greater than that of the aqueous extracts (between 10% and 18%). The highest yield recorded was in the presence of the methanolic leaf extract, followed by the stem and root extracts. Methanolic extracts are richer in polyphenols than aqueous extracts. Indeed, the highest content was observed in the leaf methanolic extract (28.4 mg GAE/g), followed by the stem (2.96 mg GAE/g), and then the root (2.62 mg GAE/g). HPLC revealed variability in the chemical compositions of the different methanolic extracts. The leaf extract was richer in phenolic acids and flavonoids than the stem and root extracts. Regarding antimicrobial activity, it was concluded that the different methanolic extracts of lentisk were more active than the aqueous extracts. In addition, the different methanolic extracts exhibited antimicrobial activity against all tested strains, despite their morphology and Gram-staining. Indeed, the maximum inhibition zones and the minimum inhibitory concentrations for the bacterial strains sensitive to the different methanolic extracts of the mastic tree were in the range of 7 to 11 mm and 12.5 to 25 µg/mL, respectively. In addition, these extracts showed greater inhibitory activity against the tested fungal strains (A. nigerandA. flavus) and yeast (C. albicans) than against the bacterial strains. These extracts can be used to treat antimicrobial infections and as food preservatives.