Research on mesophotic reefs has increased thanks to technological advances such as remotely operated vehicles (ROV). The aim of this study was to evaluate differences in ichthyofaunal assemblages between shallow (<30 m) and mesophotic (>30 m) reef zones in three sites of the Parque Nacional zona marina del Archipiélago de Espíritu Santo (PNZMAES), a Marine protected area from the Gulf of California. To accomplish this objective, we used 14 video transects recorded with an ROV in October 2019 between 13 and 72 m depth. From these recordings, we determined species richness and abundance to analyze community structure and estimate biomass. A permutational multivariate analysis of variance was used to analyze differences between zones, sites, and their interaction. The abundance and estimated biomass of trophic groups were plotted to describe the trophic structure of the depth zones at PNZMAES. We also analyzed functional structure, functional richness (FRic), functional evenness (FEve), functional divergence (FDiv), functional specialization (FSpe), and functional originality (FOri) by depth zone and video transect based on six biological traits and estimated biomass. The taxonomic and functional indices were the response variables of linear models, while the explanatory variables were in situ temperature, light, and dissolved oxygen. At PNZMAES, 61 species were registered: 42 species in the shallow zone (high abundance and estimated biomass of planktivorous) and 40 species in the mesophotic zone (high abundance of planktivorous and high estimated biomass of piscivores). When comparing the ichthyofaunal community structure, differences were observed in richness, abundance, and estimated biomass associated with the depth zone, site, and the interaction of these variables. An overlap of more than 60% in the functional volume between zones was observed. Higher values of FRic, FDiv, and FSpe in the deep zone indicated that ecological functions were equivalent to those in the shallow zone. Finally, the linear models indicated an effect of environmental variables on the ecological indicators. The mesophotic reefs of PNZMAES are potential refuges for certain species (some of them endangered) and should be considered in the proposal of management strategies to protect them from future anthropogenic threats.