Plants are alternative source of natural medicines due to secondary active metabolites. Fagonia cretica extracts and Gradient High-Pressure Liquid Chromatography fractionations were checked against multidrug-resistant gastrointestinal pathogens including, Salmonella typhi, Escherichia coli and Shigella flexneri. ESI-MS/MS analysis of bioactive HPLC fractions was performed to elucidate antibacterial compounds. F. cretica extracts exhibited potential antibacterial activity. Twenty-four (24) HPLC fractions were obtained from methanol, ethanol and aqueous extracts of F. cretica. Eighteen (18) fractions showed antibacterial activity, while no activity was observed by the remaining six (6) fractions. HPLC fractions, F1 (25g ± 0.20 mm) and F2 (15f ± 0.12 mm) of aqueous extract exhibited activity against multidrug resistant GI pathogens. Gallic acid, quinic acid, cyclo-l-leu-l-pro, vidalenolone, liquirtigenin, rosmarinic acid and cerebronic acid were identified in F1 fraction of aqueous extract, while succinic acid, cyclo (l-Leul-Pro) and liquirtigenin were identified in F2 fraction of aqueous extract through ESI-MS/MS analysis. F. cretica extracts and HPLC fractions showed potential activity against MDR GI pathogens. Vidalenolone, Cyclo-1-leu-1-pro and Cerebronic acid are first time reported in F. cretica. Further characterization of bioactive compounds from F. cretica may be helpful to elucidate antibacterial therapeutic molecules.