A better understanding of trophic interactions between hardhead catfish (Ariopsis felis) and gafftopsail catfish (Bagre marinus) is crucial for developing multi-species management strategies for the northern Gulf of Mexico (GOM). These two species are often aggregated in food web models; however, limited data are available to substantiate this approach. Therefore, the present study aimed to describe the dietary habits of hardhead catfish and gafftopsail catfish using analysis of stomach contents aided by DNA barcoding. Hardhead (n = 693) and gafftopsail (n = 655) catfish were sampled in the northern GOM from 2015–2019 using both fisheries-dependent and -independent techniques. The average percent number (%N), average percent mass (%M), prey specific number (%PN), prey specific mass (%PM), and prey-specific index of relative importance (%PSIRI) were computed to quantify prey species. The stomach content analysis identified distinct differences in diet between hardhead and gafftopsail catfish. Crustaceans were the most important prey for hardhead catfish, while gafftopsail catfish showed a significantly broader dietary breadth and were primarily piscivorous. Multivariate analyses indicated that the location of capture explained the greatest amount of diet variability for both species. These findings address fundamental knowledge gaps regarding the dietary habits of hardhead and gafftopsail catfish in northern GOM ecosystems.