Sulfate reducing bacteria (SRB) can contribute to facilitating serious concrete corrosion through the production of hydrogen sulfide in sewers. Recently, free nitrous acid (FNA) was discovered as a promising antimicrobial agent to inhibit SRB activities thereby limiting hydrogen sulfide production in sewers. However, knowledge of the bacterial response to increasing levels of the antimicrobial agent is unknown. Here we report the proteomic response of Desulfovibrio vulgaris Hildenborough and reveal that the antimicrobial effect of FNA is multi-targeted and dependent on the FNA levels. This was achieved using a sequential window acquisition of all theoretical mass spectrometry analysis to determine protein abundance variations in D. vulgaris during exposure to different FNA concentrations. When exposed to 1.0 μg N/L FNA, nitrite reduction (nitrite reductase) related proteins and nitrosative stress related proteins, including the hybrid cluster protein, showed distinct increased abundances. When exposed to 4.0 and 8.0 μg N/L FNA, increased abundance was detected for proteins putatively involved in nitrite reduction. Abundance of proteins involved in the sulfate reduction pathway (from adenylylphophosulfate to sulfite) and lactate oxidation pathway (from pyruvate to acetate) were initially inhibited in response to FNA at 8 h incubation, and then recovered at 12 h incubation. Lowered ribosomal protein abundance in D. vulgaris was detected, however, total cellular protein levels were mostly constant in the presence or absence of FNA. In addition, this study indicates that proteins coded by genes DVU2543, DVU0772, and DVU3212 potentially participate in resisting oxidative stress with FNA exposure. These findings share new insights for understanding the dynamic responses of D. vulgaris to FNA and could be useful to guide and improve the practical applications of FNA-based technologies for control of sewer corrosion.