Feeding ecology of organisms associated with floating Sargassum in the northwestern Gulf of Mexico was assessed using fatty acids. Nineteen groups were collected from the Sargassum community including four autotrophs, eight invertebrates, five juvenile fishes, and two adult fishes. Spatial and temporal variability in polyunsaturated fatty acid (PUFA) signatures of selected taxa (Sargassum fluitans [autotroph], Leander tenuicornis [primary heterotroph], Balistes capriscus [secondary heterotroph]) was examined to quantify natural variation within these dietary tracers. Although PUFA signatures varied seasonally for all three taxa, no differences were detected between samples collected in year 2000 and 2001 or from different sample locations in the northwest Gulf. PUFA signatures made up 16.3–62.3% of the total fatty acid composition of main autotrophs present in the pelagic environment [particulate organic matter (POM), epiphytic algae, S. fluitans, S. natans], and PUFA profiles of selected primary producers were distinct. Specifically, levels of 20:5 n-3, 22:5 n-3, and 22:6 n-3 were significantly higher in POM than Sargassum spp. or epiphytic algae (Cladophora sp.). Dominant PUFA in the tissue of invertebrate and vertebrate consumers were 18:2 n-6, 20:4 n-6, 20:5 n-3, 22:5n-3, 22:6 n-3 and multivariate analyses indicated that PUFA signatures of all consumers were highly similar to POM. As a result, heterotrophs utilizing the Sargassum complex may rely heavily on phytoplankton production rather than production by Sargassum or associated epiphytic algae.