In this study, inhibition of Escherichia coli glucuronidase (GUS) by a plant extract of Pistacia terebinthus was investigated. P. terebinthus (from Karaburun region in Turkey) leaf extract exhibits various bioactivities, such as antioxidant, antimicrobial and cytotoxic property because of its flavonoid, phenolic and alkaloid contents. HPLC analyses were performed to evaluate the composition of the extract and main flavonoid compounds such as luteolin, luteolin-7-glucoside and apigenin-7-glucoside were detected in extract. GUS activity and kinetic data of the GUS were obtained by measuring rate of production of resulting fluorescent 4-methylumbelliferone (4-MU) and phenolphthalein fluorometrically and spectrophotometrically, respectively. Plant leaves showed higher antioxidant capacity as 85 TEAC (trolox equivalent antioxidant capacity) value and higher antimicrobial activity towards Staphylococcus aureus. 92.4% GUS enzyme inhibition was achieved with 8.3 μg ml −1 crude extract concentration, where IC 50 value was found as 2.1 (±0.2) μg ml −1. Kinetic studies by nonlinear regression revealed that inhibition type caused by plant extract was mixed type (noncompetitive and competitive together). K m and V max values were 96.94 ± 9.68 nM and 7.81 ± 0.16 nmol min −1 mg −1, respectively. Inhibition constant ( αK i ) was found as 7.00 ± 0.93 nM having α value 2.17 ± 0.93. Our results may provide useful information regarding the inhibition of microbial GUS activity, preventing deglucuronidation and reducing possible cancer risk by bioactive P. terebinthus crude extract.