Bacterial biofilms form when bacteria adhere to a surface and produce an exopolysaccharide matrix ( Costerton Science 1999 , 284 , 1318 ; Davies Science 1998 , 280 , 295 ; Flemming Nat. Rev. Microbiol. 2010 , 8 , 623 ). Because biofilms are resistant to antibiotics, they are problematic in many aspects of human health and welfare, causing, for instance, persistent fouling of medical implants such as catheters and artificial joints ( Brunetto Chimia 2008 , 62 , 249 ). They are responsible for chronic infections in the lungs of cystic fibrosis patients and in open wounds, such as those associated with burns and diabetes. They are also a major contributor to hospital-acquired infections ( Sievert Infec. Control Hosp. Epidemiol. 2013 , 34 , 1 ; Tatterson Front. Biosci. 2001 , 6 , D890 ). It has been hypothesized that effective methods of biofilm control will have widespread application ( Landini Appl. Microbiol. Biotechnol. 2010 , 86 , 813 ). A promising strategy is to target the mechanisms that drive biofilm dispersal, because dispersal results in biofilm removal and in the restoration of antibiotic sensitivity. First documented in Nitrosomonas europaea ( Schmidt J. Bacteriol. 2004 , 186 , 2781 ) and the cystic fibrosis-associated pathogen Pseudomonas aeruginosa ( Barraud J. Bacteriol. 2006 , 188 , 7344 ; J. Bacteriol. 2009 , 191 , 7333 ), regulation of biofilm formation by nanomolar levels of the diatomic gas nitric oxide (NO) has now been documented in numerous bacteria ( Barraud Microb. Biotechnol. 2009 , 2 , 370 ; McDougald Nat. Rev. Microbiol. 2012 , 10 , 39 ; Arora Biochemistry 2015 , 54 , 3717 ; Barraud Curr. Pharm. Des. 2015 , 21 , 31 ). NO-mediated pathways are, therefore, promising candidates for biofilm regulation. Characterization of the NO sensors and NO-regulated signaling pathways should allow for rational manipulation of these pathways for therapeutic applications. Several laboratories, including our own, have shown that a class of NO sensors called H-NOX (heme-nitric oxide or oxygen binding domain) affects biofilm formation by regulating intracellular cyclic di-GMP concentrations and quorum sensing ( Arora Biochemistry 2015 , 54 , 3717 ; Plate Trends Biochem. Sci. 2013 , 38 , 566 ; Nisbett Biochemistry 2016 , 55 , 4873 ). Many bacteria that respond to NO do not encode an hnoX gene, however. My laboratory has now discovered an additional family of bacterial NO sensors, called NosP (nitric oxide sensing protein). Importantly, NosP domains are widely conserved in bacteria, especially Gram-negative bacteria, where they are encoded as fusions with or in close chromosomal proximity to histidine kinases or cyclic di-GMP synthesis or phosphodiesterase enzyme, consistent with signaling. In this Account, we briefly review NO and H-NOX signaling in bacterial biofilms, describe our discovery of the NosP family, and provide support for its role in biofilm regulation in Pseudomonas aeruginosa, Vibrio cholerae, Legionella pneumophila, and Shewanella oneidensis.