Mesopelagic fishes represent an important component of the marine food web due to their global distributions, high abundances and ability to transport organic material throughout a large part of the water column. This study combined stable isotope (SIAs) and gut content analyses (GCAs) to characterize the trophic structure of mesopelagic fishes in the North-Central Gulf of Mexico. Additionally, this study examined whether mesopelagic fishes utilized chemosynthetic energy from cold seeps. Specimens were collected (9–25 August 2007) over three deep (>1,000 m) cold seeps at discrete depths (surface to 1,503 m) over the diurnal cycle. GCA classified 31 species (five families) of mesopelagic fishes into five feeding guilds: piscivores, large crustacean consumers, copepod consumers, generalists and mixed zooplanktivores. However, these guilds were less clearly defined based on stable isotope mixing model (MixSIAR) results, suggesting diets may be more mixed over longer time periods (weeks–months) and across co-occurring species. Copepods were likely important for the majority of mesopelagic fishes, consistent with GCA (this study) and previous literature. MixSIAR results also identified non-crustacean prey items, including salps and pteropods, as potentially important prey items for mesopelagic fishes, including those fishes not analysed in GCA (Sternoptyx spp. and Melamphaidae). Salps and other soft-bodied species are often missed in GCAs. Mesopelagic fishes had δ¹³C results consistent with particulate organic matter serving as the baseline organic carbon source, fueling up to three trophic levels. Fishes that undergo diel vertical migration were depleted in ¹⁵N relative to weak migrators, consistent with depth-specific isotope trends in sources and consumers, and assimilation of ¹⁵N-depleted organic matter in surface waters. Linear correlations between fish size and δ¹⁵N values suggested ontogenetic changes in fish diets for several species. While there was no direct measure of mesopelagic fishes assimilating chemosynthetic material, detection of infrequent consumption of this food resource may be hindered by the assimilation of isotopically enriched photosynthetic organic matter. By utilizing multiple dietary metrics (e.g. GCA, δ¹³C, δ¹⁵N, MixSIAR), this study better defined the trophic structure of mesopelagic fishes and allowed for insights on feeding, ultimately providing useful baseline information from which to track mesopelagic trophodynamics over time and space.