The lined seahorse (Hippocampus erectus) is an aquacultural species in China and has important economic and ornamental value. However, the species is affected by disease, which restricts their large-scale cultivation. In 2021, a disease was observed in cultured seahorses in Tianjin, China, with a cumulative mortality rate of 20%. The symptoms observed in the moribund seahorse included a weak swimming capacity, discolored body surface, enlarged liver and kidneys, and numerous white nodules in the parenchymatous organs. The strains HM-2021-1 and HM-2021-2 were isolated from diseased seahorses and were identified as being responsible for the disease. It demonstrated the potential to infect seahorse, and the cumulative mortalities of the seahorses artificially infected with strains HM-2021-1 and HM-2021-2 were 93.3% and 90.0%. The pathogen was identified as Mycobacterium chelonae based on physiological and biochemical tests, and 16S rDNA, rpoB, and Hsp65 gene sequence analysis. To our knowledge, this study is the first report of M. chelonae associated with diseased seahorses in China.