Pathogenic bacteria that infect the gastrointestinal and respiratory tract form biofilms that are largely anaerobic, modulating their metabolic lifestyle and virulence. Many of these microbes utilize acetate fermentation via the enzyme pyruvate formate lyase (PFL), which catalyzes the production of acetyl-CoA and formate from pyruvate via a radical mechanism, enabling additional ATP production and the de novo synthesis of proteins and nucleic acids. We have investigated the mechanism of PFL via mechanism-based inhibitors to reveal the essential role of two cysteine thiyl radicals in catalysis and estimate their reactivity for the first time. Additionally, we have demonstrated the role of nitric oxide (NO) as an irreversible inhibitor of they glycyl radical enzyme PFL as well as glycyl radical enzymes as a class and their radical S-adenosylmethionine-dependent activases via the production of dinitrosyl iron complexes. These results set the stage for the development of mechanism-based inhibitors of PFL and other glycyl radical enzymes and provide a biochemically informed approach to narrow-spectrum site-specific antibiotic development for the treatment of gastrointestinal and pulmonary disease.