A key issue in the treatment of disease caused by slow-growing nontuberculous mycobacteria is the limited association between in vitro minimum inhibitory concentrations (MICs) of rifampicin and ethambutol alone and the in vivo outcome of treatment with these drugs. Combined susceptibility testing to rifampicin and ethambutol could provide a more realistic view of the efficacy of these drugs. In this study, Mycobacterium avium (n=5), Mycobacterium chimaera (n=6), Mycobacterium intracellulare (n=4), Mycobacterium xenopi (n=4), Mycobacterium malmoense (n=3) and Mycobacterium simiae (n=2) clinical isolates were selected and the MICs of rifampicin and ethambutol alone and in combination were measured using the Middlebrook 7H10 agar dilution method. Synergy was defined as a fractional inhibitory concentration index =0.5. Rifampicin and ethambutol showed synergistic activity against the majority of M. avium (4/5), M. chimaera (5/6) and M. intracellulare (3/4) isolates and 1 of 2 eligible M. malmoense isolates. No synergistic activity was measured against M. xenopi and M. simiae. Synergy was neither universal for all species nor for all isolates of one species; it thus needs to be tested for rather than assumed. Even if this synergy exists in vivo, it is questionable whether the MICs to the combined drugs can be overcome by the drug exposure attained by current regimens at the recommended dosages. New dosing strategies for rifampicin and ethambutol should be studied to increase the exposure to these drugs and thus maximise their impact.