Nitric oxide (NO) is a potent inhibitor of diverse cellular processes in bacteria. Therefore, it was surprising to discover that several bacterial species, primarily Gram-positive organisms, harboured a gene encoding nitric oxide synthase (NOS). Recent attempts to characterize bacterial NOS (bNOS) have resulted in the discovery of structural features that may allow it to function as a NO dioxygenase and produce nitrate in addition to NO. Consistent with this characterization, investigations into the biological function of bNOS have also emphasized a role for NOS-dependent nitrate and nitrite production in aerobic and microaerobic respiration. In this review, we aim to compare, contrast, and summarize the structure, biochemistry, and biological role of bNOS with mammalian NOS and discuss how recent advances in our understanding of bNOS have enabled efforts at designing inhibitors against it.