Vachellia farnesiana is a medicinal plant rich in bioactive compounds, long used in traditional medicine. Its therapeutic potential includes addressing cancer, diabetes, and antibiotic-resistant infections, making it a promising candidate for drug discovery. This study focused on isolating active compounds from the n-hexane and dichloromethane (DCM) fractions of the plant's ethanolic extract employing size exclusion chromatography and nuclear magnetic resonance (NMR) spectroscopy. It also aimed to assess their potential antimicrobial, antidiabetic, and cytotoxic activities while validating traditional uses through experimental and computational approaches. The biological activities of these compounds were tested through cytotoxicity and antimicrobial assays, glucose-lowering experiments in mice, and molecular docking to simulate interactions with disease-related proteins. Five compounds were isolated, including β-amyrin, 7β-hydroxysitosterol, oleanolic acid, lauric acid, and viscic acid. Notable findings include high cytotoxicity (LC50 = 0.231 µg/mL for the n-hexane fraction), antimicrobial activity with inhibition zones reaching 19 mm, and a 57.53% reduction in blood glucose levels (DCM fraction, 400 mg/kg, 3 h). Molecular docking demonstrated strong binding affinities for the isolated compounds against targeted receptors. This research validates V. farnesiana's traditional medicinal uses and emphasizes its potential as a source of novel treatments for chronic diseases and infections and paves the way for future research.