A multidisciplinary approach including biometric, ecological, and molecular genetic analyses was employed to test species status of the Black Moray Muraena augusti (Kaup, 1856) and the Mediterranean Moray M. helena Linnaeus, 1758. Relevant differences were identified in their habitat, bathymetric range, color pattern, vertebral formula, growth parameters and lifespan, reproduction strategy, size/age at first maturity, and distribution. Intra- and interspecific genetic divergences (based on a region of the rhodopsin gene and the complete cytochrome b gene) also suggest that both moray eels are different species. Mitochondrial and nuclear sequence data place these two species into different clades. Phylogenetic analyses among an additional five species of moray eels occurring in sympatry in the eastern central Atlantic resolved M. helena, M. augusti, and M. melanotis as closely related species in a well supported clade, while M. robusta emerged as a more divergent species within the Gymnothorax clade. Based on these findings, Muraena augusti is a valid species and should be resurrected.