Sympatric species that share morphological similarities tend to diversify their prey resources to coexist. The striped cowfish (Aracana aurita) and ornate cowfish (A. ornata) live together on the continental shelf from western to southern Australia including Tasmanian waters. The present study analyzed stomach contents and stable isotope ratios (δ13C and δ15N) to investigate the use of food resource between the two species collected in Tasmanian waters, Australia. In total, 139 A. aurita and 46 A. ornata were collected from 2014 to 2018. The small size class of A. aurita (<140 mm SL) mainly consumed polychaetes and amphipods, while its large size class (≥140 mm SL) preyed more on bivalves and crustacean decapods. The main prey items of the small size class of A. ornata (<140 mm SL) were amphipods and crabs. No differences in diet were observed between the sexes of either species. Stable isotope analyses showed significant differences in δ15N and δ13C between the two species, but not between the two size classes of A. aurita (ANOVA, P < 0.05). Integrating analyses of stomach contents and stable isotopes indicated that the two species preferred different food resources based on different trophic sources. Consequently, although the habitats of the two species overlap, both species can co-exist through dietary segregation.