This study was conducted to determine the total antioxidant capacity (TAC), anti-quorum sensing potential, and anticancer activity of extracts obtained from Berberis crataegina fruits by the maceration method. Moreover, the mode of action for the antioxidant activity was explored by molecular modeling. For this purpose, we performed DPPH, FRAP, and CUPRAC methods to measure the antioxidant ability of the extracts. The total phenolic content (TPC) of the extracts was determined by the Folin-Ciocalteu method. The cytotoxic activity of the crude extracts on the A549 lung cancer cell line was evaluated using the MTT assay. The TAC values of the extract were found to vary between 0.744 and 1.763 mmol TE/g-extract for FRAP, CUPRAC, and DPPH assays. The TPC of extract was determined as 80.76 mg GAE/g. Extract induced inhibition of virulence factors elastase and pyocyanine by 85.8% and 57%, respectively. Results provided evidence that the extracts exhibited significant cytotoxicity against A549 human lung cancer cells with an IC50 value of 215.99 μg/mL (p<
0.05). Additionally, computational studies revealed that the major compounds of the extract, chlorogenic acid and caffeic acid, could bind to target enzymes and form stable complexes. These findings significantly promote the development and application of Berberis crataegina fruits.