The mesopelagic region (200–1000 m) hosts a wide variety of organisms in a concentrated layer known as the deep scattering layer (DSL). Much of the mesopelagic region in the central North Pacific remains unexplored, limiting ecosystem considerations in fisheries management and other applications. The National Oceanic and Atmospheric Administration (NOAA) Office of Ocean Exploration and Research used NOAA Ship Okeanos Explorer to conduct two expeditions in the region, one to Johnston Atoll and the other to Musicians Seamounts. During these expeditions, a remotely operated vehicle collected video and CTD environmental data while the ship collected active acoustics. This project analyzed the video, CTD, and acoustic data to create an ecological characterization of the mesopelagic at Johnston Atoll and Musicians Seamounts. The initial characterization included a full inventory of mesopelagic fauna imaged, the relationship between the abundance of categorical groups (gelatinous, Chaetognatha, Crustacea, and fish) and the environmental variables of salinity, oxygen, and temperature, a comparison between the abundance of mesopelagic fauna below, within, and above the DSL, and a qualitative analysis of the spatial distribution of categorical groups. There was a significant negative relationship between the abundance of the gelatinous organisms and salinity and oxygen concentrations. Overall, there were more sightings per minute of all organisms combined below and within the DSL than above the DSL at Musicians Seamounts, while there was no difference in sightings per minute between the below, within, and above DSL groups at Johnston Atoll. Proportionally, there were more gelatinous organisms than any other single category (fish, Chaetognatha, and Crustacea) at all dive sites in the study. These initial characterizations strive to develop biodiversity concepts and the relationships between the environment and fauna. Progress in these areas can aid the management of marine national monuments and serve as a baseline for future ROV dives.