Recent losses of seagrass beds have resulted in a decrease in seagrass-associated fishes, highlighting the need for the improved management of such habitats and resources. Analyses of feeding patterns of fish associated with seagrass habitats are useful for understanding their habitat dependence, which contributes to achieving their effective conservation. The gut contents of 61 fish species were examined to clarify the food habits of subtropical seagrass fishes in Nagura Bay, Ishigaki Island, southern Japan. A cluster analysis based on dietary overlaps showed that the seagrass fish assemblage comprised six trophic groups (small crustacean, large crustacean, detritus, plant, fish and zooplankton feeders). Small and large crustacean feeders were the most abundant, followed by detritus, plant and fish feeders. Zooplankton feeders were represented by only two species. Ontogenetic changes in food preference were recognized in four species, including Lutjanus fulviflamma, Ostorhinchus ishigakiensis, Gerres oyena and Siganus fuscescens. Small individuals of the four species generally fed on small crustaceans or detritus, subsequently switching to other larger prey items with growth (e.g. crabs, shrimps and seagrass fronds). The most important food items for the assemblage comprised harpacticoid copepods, shrimps, crabs and detritus, representing high cumulative gut volumes for all fishes. Some species showed diets remarkably different from those determined for fishes in other seagrass beds off a nearby neighbouring island, evidence of the food-use patterns of seagrass fishes varying on a small spatial scale. Such dietary flexibility may increase the adaptability of fishes to seagrass beds characterized by significant variations in available food resources.