Aims: To develop and evaluate an antimicrobial supplement for use with phage-based tests for rapid detection of drug resistance of tuberculosis (TB). Methods and Results: An antimicrobial formulation containing nystatin, oxacillin and aztreonam (NOA) (final concentrations of 50 000 IU l(-1), 2 mg l(-1), and 30 mg l(-1) respectively) was developed. This formulation was tested for its influence on detection of a number of Mycobacterium tuberculosis (MTB) strains using the phage amplification (FASTPlaque) assay. Addition of the supplement did not lead to significant reduction in assay sensitivity. Antimicrobial efficacy was assessed with a range of Gram-positive and -negative organisms. The NOA supplement had a broad antimicrobial effect. The supplement was tested for its effect on growth of MTB culture, and on determination of rifampicin resistance using the phage-based methodology (FASTPlaque-Response). NOA did not significantly affect the growth of a range of rifampicin susceptible and resistant MTB strains, nor did it have an adverse effect on the number of interpretable results, nor the ability to discriminate between rifampicin susceptibility and resistance. Conclusion, Significance and Impact of the Study: Use of NOA antimicrobial supplement with rapid phage-based tests for TB will increase the proportion of interpretable results obtained, and enable their wider implementation in disease-endemic countries by improved control of specimen-related contamination.