Neisseria gonorrhoeae is the etiologic agent of gonorrhea, which has been among the most frequently reported communicable diseases in the United States since 1960. Women frequently do not exhibit symptoms, which can lead to chronic infection. N. gonorrhoeae readily forms biofilms over abiotic surfaces, over primary and transformed cervical epithelial cells, and over cervical tissues in vivo. Biofilms are often associated with chronic infection, which suggests a link between biofilm formation and asymptomatic gonorrhea in women. Proteins involved in anaerobic metabolism and oxidative-stress tolerance are critical for normal biofilm formation of N. gonorrhoeae. Therefore, we examined the spatial profiles of anaerobic respiration in N. gonorrhoeae, using an aniA - gfp transcriptional fusion. Nitric oxide (NO) can elicit biofilm dispersal when present at sublethal concentrations in the surrounding medium. Some reports indicate that NO may also encourage biofilm formation at higher, potentially lethal concentrations. NO is produced by polymorphonuclear lymphocytes (PMNs) and cervical endothelial and epithelial cells. Thus, we also examined the effect of NO on N. gonorrhoeae biofilms. We found that anaerobic respiration occurs predominantly in the substratum of gonococcal biofilms and that expression of aniA is induced over time in biofilms. Treatment with high concentrations of a rapid-release NO donor prevents biofilm formation when supplied early in biofilm development but can also enhance biofilm formation once anaerobic respiration is initiated. NO treatment partially restores biofilm formation in an aniA::kan insertion mutant, which suggests that N. gonorrhoeae in biofilms may use NO as a substrate for anaerobic growth but prefer nitrite.