Summary: Nitric oxide (NO) is an important defense molecule secreted by the squid Euprymna scolopes and sensed by the bacterial symbiont, Vibrio fischeri, via the NO sensor HnoX. HnoX inhibits colonization through an unknown mechanism. The genomic location of hnoX adjacent to hahK, a recently identified positive regulator of biofilm formation, suggested that HnoX may inhibit colonization by controlling biofilm formation, a key early step in colonization. Indeed, the deletion of hnoX resulted in early biofilm formation in vitro, an effect that was dependent on HahK and its putative phosphotransfer residues. An allele of hnoX that encodes a protein with increased activity severely delayed wrinkled colony formation. Control occurred at the level of transcription of the syp genes, which produce the polysaccharide matrix component. The addition of NO abrogated biofilm formation and diminished syp transcription, effects that required HnoX. Finally, an hnoX mutant formed larger symbiotic biofilms. This work has thus uncovered a host‐relevant signal controlling biofilm and a mechanism for the inhibition of biofilm formation by V. fischeri. The study of V. fischeri HnoX permits us to understand not only host‐associated biofilm mechanisms, but also the function of HnoX domain proteins as regulators of important bacterial processes. Biofilm formation by Vibrio fischeri is positively controlled by the sensor kinase HahK, which activates transcription of the syp locus, resulting in the production of the major polysaccharide component of the matrix and permitting cell aggregation phenotypes. HahK is negatively regulated by the nitric oxide‐binding protein HnoX. Thus, presence of nitric oxide controls biofilm formation by negatively controlling the activities of HnoX and thus HahK, ultimately impacting syp transcription and production of matrix polysaccharide. [ABSTRACT FROM AUTHOR]