There is great potential to use free nitrous acid (FNA/HNO2), the protonated form of nitrite, as an antimicrobial agent due to its bacteriostatic and bactericidal effects on a range of microorganisms. Here, we determine the effects of FNA on the opportunistic pathogen Pseudomonas aeruginosa PAO1, a well-studied denitrifier capable of nitrate/nitrite reduction in its anaerobic respiration. It was seen that lower FNA concentrations in the range of 0.1 to 0.2 mg N/L exerted a temporary inhibitory effect on the growth of P. aeruginosa, while respiratory inhibition was not detected until an FNA concentration of 1.0 mg N/L was applied. The FNA concentration of 5.0 mg N/L caused complete cell killing and likely cell lysis. The results suggest concentration-related and multiple antimicrobial effects of FNA. Differential killing of FNA in the P. aeruginosa subpopulations was detected, suggesting intrastrain heterogeneity, and does not support the idea of specific concentrations of FNA bringing about bacteriostatic and bactericidal effects on this species. A delayed recovery from FNA treatment suggested that FNA caused cell damage which required repair prior to the organism showing cell growth. The results of the study provide insight of the inhibitory and biocidal mechanisms of FNA on this important microorganism.