Floating macroalgal rafts can form extensive habitat in coastal waters and support abundant and diverse communities of juvenile fishes. Rafts comprised predominantly of Sargassum spp. are common within the Ningaloo Coast World Heritage Area, Western Australia, and may be an important transitional habitat for pre-settlement coastal fishes. We had three aims: 1) to quantify the diversity and abundance of fishes associated with Sargassum rafts; 2) to use stable isotopes to determine whether fishes associating with rafts preyed on nearby plankton or on prey associated with Sargassum; and 3) to determine whether the food web fishes relied upon was ultimately supported by primary production of Sargassum or planktonic phytoplankton (measured as seston). Fishes and macroinvertebrates associating with Sargassum rafts, zooplankton, and seston were collected at six sites in the Ningaloo Marine Park and analysed for δ15N and δ13C, and the diets of the most common fishes were analysed using MixSIAR. Fishes were more abundant around macroalgal rafts than in open water. Eleven species of juvenile fishes associated with Sargassum rafts, with one species (Histrio histrio (Antennariidae)) represented by juveniles and adults. Four of the five fishes most sampled around Sargassum spp. rafts, (Abudefduf vaigiensis (Pomacentridae), Helotes sexlineatus (Terapontidae), Colurodontis paxmani (Monacanthidae) and Petroscirtes breviceps (Blenniidae)) had generalist diets whose food webs were ultimately supported by greater contributions of primary production from Sargassum spp. (55–72%) than planktonic seston. H. histrio was predominantly piscivorous but still derived most (64%) of its organic matter indirectly from Sargassum spp. Our results reveal the importance of Sargassum rafts as habitat that provides shelter and food for juvenile fishes and adult H. histrio. Moreover, the prevalence of juvenile fishes associating with Sargassum rafts highlights a bidirectional form of benthic-pelagic coupling whereby macroalgae that detach from the benthos provide habitat and a trophic subsidy for pelagic juvenile fishes before they the transition into benthic coastal habitats.