Hydrogen sulfide produced by sulfate reducing bacteria (SRB) in sewers causes odor problems and asset deterioration due to the sulfide induced concrete corrosion. Free nitrous acid (FNA) was recently demonstrated as a promising antimicrobial agent to alleviate hydrogen sulfide production in sewers. However, knowledge of the antimicrobial mechanisms of FNA is largely unknown. Here we report the multiple-targeted antimicrobial effects of FNA on the SRB Desulfovibrio vulgaris Hildenborough by determining growth, physiological and gene expression responses to FNA exposure. The activities of growth, respiration and ATP generation were inhibited when exposed to FNA. These changes were reflected in transcript levels detected during exposure. Removal of FNA was evident by nitrite reduction that likely involved nitrite reductase and the poorly characterised hybrid cluster protein, and the genes coding for these proteins were highly expressed. During FNA exposure lowered ribosome activity and protein production were detected. Additionally, conditions within the cells were more oxidising and there was evidence of oxidative stress. Based on interpretation of the measured responses we present a model depicting the antimicrobial effects of FNA on D. vulgaris. These findings provide new insight for understanding the responses of D. vulgaris to FNA and will provide foundation for optimal application of this antimicrobial agent for improved control of sewer corrosion and odor management.