Background: Antimicrobial resistance is a major threat to humankind and the advancement of resistance due to genetic modifications and other defense mechanisms that make the current antibiotics ineffective or less efficacious. Objective: This investigation aims to isolate bioactive compounds from the leaf acetone extract of Terminalia catappa and to evaluate their antimicrobial potential against human pathogenic organisms. Materials and Methods: The bioactive extract was subjected to column chromatography. The fractions were assessed for their minimum inhibitory concentration, minimum fungicidal concentration, and time kill assays. UHPLC-MS/MS analysis was used to identify the bioactive molecules in the fraction. Results: The isolated fraction exhibited antimicrobial activity, with the most sensitive being Staphylococcus aureus (clinical isolate) and Methicillin Resistant Staphylococcus aureus 1503 (0.097 mg/mL), and the fungi Trichophyton rubrum and Candida albicans were inhibited at 0.097 mg/mL. The time kill assay exhibited bactericidal properties towards S. aureus (clinical isolate) and Salmonella typhi (MTCC 733). Additionally, MRSA 1503 and Proteus vulgaris exhibited bacteriostatic activities. The UHPLC-MS/MS analysis revealed that the fraction was rich in polyphenols. Alkaloids and some ellagitannins were identified for the first time. Conclusion: The results highlight the significant inhibition of multidrug-resistant MRSA strains and fungi by the polyphenol-rich fraction. The investigation reveals the potential use of the identified compounds for antimicrobial use, which could lower the implication of multidrug resistance. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.