Despite its extreme geographical isolation, numerous expeditions have surveyed the marine flora and fauna of Johnston Atoll. However, historical information about the marine biodiversity of Johnston is mostly limited to SCUBA surveys in shallow-waters (<30 m), and submersible observations in deeper waters (100–500 m). Extensive coral reefs, known as mesophotic coral ecosystems, exist between these two depth ranges at Johnston, but have remained largely unexplored. We used closed-circuit rebreathers to survey eleven sites at mesophotic depths (32–78 m) surrounding Johnston Atoll. A total of 130 species were recorded, including 99 species of fish, 15 species of corals, nine species of macroalgae, three species of echinoderms, three species of sponges and one species of squat lobster. Most species recorded during our mesophotic surveys have previously been recorded on shallow-water (<30 m) reefs at Johnston, with the exception of one black coral, one zoanthid, one squat lobster, two macroalgae, three sponges, and 22 fish, which represent new records for the atoll. As noted in previous studies, our surveys found a near absence of endemism, and recorded high proportions of species that are also known from the Hawaiian Archipelago. The similarity between the mesophotic biodiversity of Johnston Atoll and Hawai‘i provides further support for the strong connectivity between these two locations highlighted in previous studies.