Holacanthus ciliaris is an important benthic-feeding reef fish but the relationship between the composition of its diet and prey availability is still unknown. Here we determined the quantitative composition of the gut contents of H. ciliaris in São Pedro e São Paulo Archipelago, Brazil (SPSPA), and compared it to the abundance of benthic organisms in the area. Holacanthus ciliaris has a relatively diversified diet with more than 30 prey species in SPSPA, especially sponges (13 spp., average 68% of gut contents total weight), algae (12 spp., 25%) and bryozoans (3 spp., 5%). In contrast, the benthic community is composed mainly of algae (average 81% of total cover) and followed by sponges (13%), bryozoans (5%), cnidarians (0.5%), polychaetes (0.5%) and tunicates (0.5%). The most common species were the algae Caulerpella ambigua and Caulerpa racemosa var. peltata; the bryozoan Margaretta buski; and the sponges Scopalina ruetzleri, Chondrosia collectrix and Clathria calla. The Manly resource selection function showed that H. ciliaris preferred the sponges Geodia neptuni, Erylus latens, Clathria calla and Asteropus niger, among others, and avoided common species such as the sponges Scopalina ruetzleri, Dysidea etheria and Hemimycale insularis and the algae Caulerpella ambigua, Bryopsis plumosa and Neomeris annulata. Kendall's rank correlation index showed no significant correlation between prey abundance in the field and in the diet of H. ciliaris, which seems to actively choose relatively rare and less defended prey.