The escalating crisis of antimicrobial resistance (AMR) has necessitated innovative approaches to antibiotic discovery, with natural products emerging as promising candidates due to their structural diversity and unique mechanisms of action. This study aimed to evaluate the antimicrobial potential of natural product extracts derived from underexplored environmental niches against multidrug-resistant pathogens. Samples from soil, marine sediments, and endophytic microorganisms were collected, processed, and screened using agar diffusion and broth microdilution assays to determine their antimicrobial activity. The most active extracts were fractionated using high-performance liquid chromatography (HPLC), and their bioactive compounds were characterized through nuclear magnetic resonance (NMR) and mass spectrometry (MS).

Results indicated that Extract_2 exhibited the highest activity, particularly against Staphylococcus aureus, with inhibition zones averaging 19 mm. However, its efficacy was generally lower than standard antibiotics such as Vancomycin and Ciprofloxacin, which achieved inhibition zones of 24–27 mm. Statistical analysis revealed a significant difference in activity for S. aureus (p = 0.030) but not for other pathogens. These findings highlight the potential of natural products as a basis for new antibiotics, while also underscoring the need for optimization to enhance their potency and specificity. The study recommends expanding the search for bioactive compounds in extreme environments, employing advanced screening technologies, and exploring synergistic combinations with existing antibiotics.