METHODS Damien Foundation Project, Bangladesh. OBJECTIVE To evaluate sputum smear fluorescein diacetate (FDA) vital staining to predict culture-defined failure and rifampicin (RMP) resistance. METHODS A retrospective, operational study. RESULTS A total of 1633 episodes of auramine smear-defined late conversion and failure could be evaluated (respectively 640 and 584 on first treatment and 185 and 224 on retreatment). Negative FDA was 95% predictive of negative culture in patients on first treatment, while its positive predictive value was around 95% during retreatment. The predictive value of a positive (not scanty) result for RMP resistance or environmental non-tuberculous mycobacteria (NTM) was at least 90%, except in late converters on first-line treatment; a negative result was over 95% exclusive of the same except in retreatment failures. FDA correctly identified 88-98% of all RMP resistance. CONCLUSIONS FDA staining increased the proportion of tuberculosis patients put on second-line treatment without receiving the standard first-line retreatment regimen. In our setting, with excellent microscopy, late case presentation and low resistance prevalence, it proved indispensable for efficient culture and referrals of early suspects for rapid drug susceptibility testing (DST). In other settings with low prevalence of NTM and difficult access to accurate and rapid DST, FDA-positive failures might even be considered for immediate start of second-line treatment.