Background/Objectives: The rise in antimicrobial resistance is one of the major challenges to global health systems, which necessitates the development of new antibacterial compounds. The bioactive compounds of brown seaweed Sargassum duplicatum have demonstrated potential antibacterial activity. This study applied metabolomic profiling and molecular networking in combination with antibacterial screening assays to assess the antimicrobial properties of S. duplicatum extracts against multidrug-resistant bacteria. Methods: Two extraction methods, i.e., maceration and microwave extraction, were used. Therewith, untargeted metabolomic profiling was performed using Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS). Molecular networks (MNs) were established and compound dereplication was conducted using the spectral database of the Global Natural Products Social Molecular Networking platform (GNPS). Additionally, antimicrobial assays were conducted against Gram-positive and Gram-negative bacterial strains, including multidrug-resistant bacteria, i.e., methicillin-resistant Staphyloccocus aureus ATCC 33592 (MRSA) and β-lactamase, producing Escherichia coli ATCC 35218 (TEM-1 positive strain). Result: Dereplication resulted in the prediction of six compounds with reported antimicrobial properties, i.e., 13-docosenamide, 9-octadecenamide, pheophorbide A, ouabain, sarmentoside B and AC1L1X1Z. Antibacterial screening of the extracts revealed that the ethyl acetate maceration extracts exhibited the strongest inhibitory activity, with inhibition values between 85 and 98% against S. aureus ATCC 33592. Conclusions: This metabolomics study requires further research to isolate, purify, confirm, and validate the dereplicated compounds that may have potential antibacterial activity.

Sargassum is a widespread brown seaweed species and a source of bioactive compounds with promising antioxidant potential. Unfortunately, to date, the Sargassum species remains largely unexplored. This study was conducted to explore the bioactive compounds from Sargassum ilicifolium extracts collected from Nguyahan and Sundak Beaches, Gunungkidul, Indonesia, by observing Liquid Chromatography–High Resolution Mass Spectrometry (LC-HRMS)-based metabolomics profiling and antioxidant activity assays. Metabolomic analysis detected 506 molecular features across different extraction methods and solvents, with five metabolites putatively dereplicated, including atractylenolide III, pheophorbide A, 13-docosenamide, 1,3,6,8-tetrahydroxy-2-(1-hydroxyhexyl)anthracene-9,10-dione, and 5-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid. Extraction parameters, particularly solvent polarity and sample pretreatment, have been shown to affect the metabolite variation. Dried samples showed less variation in metabolites than the fresh sample. Antioxidant activity assay showed a moderate to high radical scavenging activity (30–100%), with methanol extracts as a polar solvent inhibited more than semipolar solvents. This study provides a metabolomics-guided assessment of the antioxidant potential of S. ilicifolium, supporting its value and potential as a source of bioactive compounds for future pharmaceutical and nutraceutical applications.