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 (SIA) and gut content analyses (GCA) 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 (>1000 m) cold seeps at discrete depths (surface to 1503 m) over the diurnal cycle. Gut content analyses classified 31 species (5 families) of mesopelagic fishes into 5 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) and across co-occurring species. Euphausiids 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 analyzed in GCA (Sternoptyx spp. and Melamphaidae). Salps and other soft-bodied species are often missed in gut content analyses. Mesopelagic fishes had δ13C results consistent with particulate organic matter (POM) serving as the baseline organic carbon source, fueling up to 3 trophic levels. Fishes that undergo diel vertical migration were depleted in 15N relative to weak migrators, suggesting depth-specific isotope patterns in sources and consumers. 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, it is possible that detection of infrequent consumption of this food resource may be hindered by the assimilation of isotopically-enriched photosynthetic based 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 trophodynamic changes over time and space.