ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are a rich source of new antibacterial leads. One such plant is Senna alata (L.) Roxb (Fabaceae), a valuable medicinal tree known in folk medicine for its effectiveness in treating various ailments such as ringworms, wounds, diabetes, skin diseases, hypertension, malaria, mycosis, and bacterial diseases. AIM OF THE STUDY: This study assesses the antibacterial and antibiotic-resistance reversal properties of S. alata leaf extracts against 32 multi-drug resistant (MDR) clinically relevant bacterial strains and clinical isolates. MATERIALS AND METHODS: The hydromethanol extract (70%) was obtained through ultrasound-assisted extraction, followed by partitioning with solvents of increasing polarity, specifically petroleum ether (PE), ethyl acetate (EA), n-butanol (n-BuOH), and water, to isolate different fractions. Antibacterial and combination tests were conducted using the broth microdilution method. Subsequently, high-resolution HPLC-ESI-QTOF-MS analysis was performed to profile the bioactive secondary metabolites in the most active fractions. In addition, the pharmacokinetic (PK) properties, drug-likeness, and medicinal chemistry of the key phytoconstituents were predicted in silico using SwissADME. Moreover, we utilized the ProTox-II web server to predict the toxicity profile of the potential drug candidates. RESULTS: The herbal fractions, except for the water fraction, showed remarkable antibacterial activity, with MICs ranging from 16 to 1024 μg/mL. The ethyl acetate (SA-EA) and n-butanol (SA-n-BuOH) fractions were the most potent, with the overall most significant effects recorded with SA-EA (with MIC <
100 μg/mL on 31 out of the 32 MDR studied strains). Additionally, SA-EA enhanced the efficacy of antibiotics, leading to up to a 64-fold reduction in MICs (of chloramphenicol, imipenem, ciprofloxacin, cefepime, and doxycycline) in combination. A total of 27 and 36 compounds were tentatively identified from SA-EA and SA-n-BuOH, respectively, with the majority being phenolic compounds known for their antibacterial properties. Furthermore, 17 phytochemicals were reported for the first time in S. alata fractions. Seven metabolites, including phloretin, 7,4'-dihydroxyflavone, isorhamnetin, apigenin, genistein, naringenin, and lactarorufin B, emerged as potential drug candidates that satisfy most of the drug candidacy criteria and PK profile amongst which apigenin, genistein, and naringenin depicted the best safety profile. CONCLUSION: The positive outcomes observed in the antibacterial activity assays, coupled with the presence of bioactive metabolites and emerging drug leads in these fractions, underscore the importance of selecting S. alata for the discovery and development of new antibacterial agents targeting MDR phenotypes.