Biofilms are surface-associated, multicellular communities of bacteria. Once established, they are extremely difficult to eradicate by antimicrobial treatment. It has been demonstrated in many species that biofilm formation may be regulated by the diatomic signaling molecule nitric oxide (NO). Although this is still a relatively new area of research, we review here the literature reporting an effect of NO on bacterial biofilm formation, emphasizing dose-dependent responses to NO concentrations when possible. Where it has been investigated, the underlying NO sensors or signaling pathways are also discussed. Most of the examples of NO-mediated biofilm regulation have been documented with exogenously applied NO, but we also survey possible natural sources of NO in biofilm regulation, including endogenously generated NO. Finally, because of the apparent broad-spectrum, antibiofilm effects of NO, NO-releasing materials and prodrugs have also been explored in this minireview.