The mode of action of the antimicrobial agent, 5-bromo-5-nitro-1,3-dioxane(bronidox), was studied in detail for gram-positive and gram-negative bacteria, yeast and fungi. Thestudies included MIC testing, thiol inhibition of activity, intracellular leakage, oxygen consumption,incorporation of 3H-uridine, scanning electron microscopy, inhibition of enzyme activity (papain) andin vitro oxidation of thiols to disulfides. It appears that the primary mode of action of bronidox isthe same as, or similar to, that of bronopol, i.e. the oxidation of essential protein thiol causing inhibitionof enzyme activity and subsequent inhibition of microbial growth.