Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen associated with healthcare-related infections that are often challenging to treat. Conditions such as, skin and soft tissue infections, bloodstream infections, and pneumonia highlight the critical need for effective therapeutic strategies. Careful use of antibiotics under medical supervision is essential to prevent the further emergence of MRSA. Recent studies have documented the antibacterial efficacy of certain endophytic fungi extracts against MRSA, suggesting their potential as a source of novel treatments. This study investigates the metabolomic profiling of the endophytic fungus Aspergillus sp. SH1 using liquid chromatography-high-resolution electrospray ionization mass spectrometry (LC-HR-ESI-MS) and evaluates the anti-MRSA potential of the fungal extract. The metabolomic analysis identified 27 compounds (1–27) with diverse chemical natures, including polyketides, alkaloids, cyclic tripeptides, polypropionate derivatives, and sesquiterpenes. The fungal extract exhibited potent anti-MRSA activity, with an IC50 value of 9.8 µg/mL, compared to ciprofloxacin (IC50 = 25.7 µg/mL). To support these findings, in silico studies were performed to model the binding interactions of the identified compounds with key MRSA-related targets, including Toll-like receptor 2 (TLR2), von Willebrand factor (VWF), tumor necrosis factor (TNF), and penicillin-binding protein 2a (PBP2a). Compounds 2, 9, 15, 16, 20, 22, and 25 demonstrated enhanced binding affinities, suggesting their potential as lead molecules for developing new antibacterial agents targeting MRSA. In conclusion, this study highlights the promising anti-MRSA potential of Aspergillus sp. SH1 extract, providing a foundation for further exploration of its bioactive compounds in combating resistant bacterial infections.