ABSTRACT: Marine organisms, including seagrasses, are important sources of biologically active molecules for the treatment of human diseases. In this study, organic extracts of the marine seagrass Halophila stipulacea obtained by different polarities from leaves (L) and stems (S) (hexane [HL, HS], ethyl acetate [EL, ES], and methanol [ML, MS]) were tested for different bioactivities. The screening comprehended the cytotoxicity activity against cancer cell lines grown as a monolayer culture or as multicellular spheroids (cancer), glucose uptake in cells (diabetes), reduction of lipid content in fatty acid-overloaded liver cells (steatosis), and lipid-reducing activity in zebrafish larvae (obesity), as well as the antifouling activity against marine bacteria (microfouling) and mussel larval settlement (macrofouling). HL, EL, HS, and ES extracts showed statistically significant cytotoxicity against cancer cell lines. The extracts did not have any significant effect on glucose uptake and on the reduction of lipids in liver cells. The EL and ML extracts reduced neutral lipid contents on the larvae of zebrafish with EC50 values of 2.2 µg/mL for EL and 1.2 µg/mL for ML. For the antifouling activity, the HS and ML extracts showed a significant inhibitory effect (p < 0.05) against the settlement of Mytilus galloprovincialis plantigrade larvae. The metabolite profiling using HR-LC-MS/MS and GNPS (The Global Natural Product Social Molecular Networking) analyses identified a variety of known primary and secondary metabolites in the extracts, along with some unreported molecules. Various compounds were detected with known activities on cancer (polyphenols: Luteolin, apeginin, matairesinol), on metabolic diseases (polyphenols: cirsimarin, spiraeoside, 2,4-dihydroxyheptadec-16-ynyl acetate; amino acids: N-acetyl-L-tyrosine), or on antifouling (fatty acids: 13-decosenamide; cinnamic acids: 3-hydroxy-4-methoxycinnamic acid, alpha-cyano-4-hydroxycinnamic), which could be, in part, responsible for the observed bioactivities. In summary, this study revealed that Halophila stipulacea is a rich source of metabolites with promising activities against obesity and biofouling and suggests that this seagrass could be useful for drug discovery in the future.