Macroalgae are valuable natural sources for bioprospection and the development of raw materials applicable to the nutrition, health, and agriculture industries. To build a basis for the sustainable use of marine organisms from the Colombian Caribbean, a preliminary study was conducted focusing on known functional compounds in two genera of macroalgae, including the species Acanthophora spicifera (Rhodophyta), Sargassum ramifolium, and Sargassum fluitans (Ochrophyta). This study included the extraction and identification of polysaccharides using ultrafiltration, nuclear magnetic resonance (¹H-NMR), Fourier-transform infrared spectroscopy (FT-IR), and size exclusion chromatography (SEC); fatty acids by gas chromatographic (GC) profiling; and phenolic composition and antioxidant activity by complementary semi-quantitative methods (ABTS, DPPH, FRAP, and ORAC assays). Carrageenan-type polysaccharides were detected in A. spicifera, while alginate and fucoidan types were found in S. ramifolium and S. fluitans; palmitic acid was the predominant fatty acid in A. spicifera and S. ramifolium, but it was not detected in S. fluitans. S. ramifolium showed the highest ABTS, DPPH, and ORAC activities and phenolic compounds, while S. fluitans exhibited the highest FRAP activity. This study contributes to the chemical knowledge on Colombian macroalgae to establish potential applications in various fields, including biomedicine, cosmetics, functional foods, and nutraceutical ingredients.