Disease due to non-tuberculous mycobacteria (NTM) is common in fish. Current recommendations focus on outbreak management by depopulating entire fish stocks and disinfecting tanks. Treatment is not advocated. Treatment may be appropriate, however, where individual, valuable fish are concerned. ZSL London Zoo managed an outbreak of mycobacteriosis in a valuable group of imported F1 captive-bred Australian lungfish (Neoceratodus fosteri) by depopulation, isolation, extensive testing and daily oral antibiotic treatment. Four species of Mycobacterium (M. marinum, M. fortuitum, M. chelonae and M. peregrinum) were involved in this outbreak, each with unique antibiotic sensitivities. Triple therapy with rifampicin, doxycycline and enrofloxacin for 8 months was the most effective antibiotic combination, resulting in full disease resolution. No side effects were noted and, more than 18 months post-treatment, no recurrence had occurred. This is the first report of mycobacterial disease in lungfish and the first report of a polymycobacterial outbreak in fish involving these four species of Mycobacterium. This report demonstrates the value of extensive isolation and identification. Also, as therapies currently advised in standard texts did not reflect the antibiotic sensitivity of the NTM found in the fish reported here, we recommend that antibiotic treatment should always be based on sensitivity testing.