Azospirillum brasilense is a rhizobacterium that provides beneficial effects on plants when they colonize roots. The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with surfaces in response to appropriate signals. Nitric oxide (NO) is a signaling molecule implicated in numerous processes in bacteria, including biofilm formation or dispersion, depending on genera and lifestyle. Azospirillum brasilense Sp245 produces NO by denitrification having a role in root growth promotion. We analyzed the role of endogenously produced NO on biofilm formation in A. brasilense Sp245 and in a periplasmic nitrate reductase mutant (napA::Tn5; Faj164) affected in NO production. Cells were statically grown in media with nitrate or ammonium as nitrogen sources and examined for biofilm formation using crystal violet and by confocal laser microscopy. Both strains formed biofilms, but the mutant produced less than half compared with the wild type in nitrate medium showing impaired nitrite production in this condition. NO measurements in biofilm confirmed lower values in the mutant strain. The addition of a NO donor showed that NO influences biofilm formation in a dose-dependent manner and reverses the mutant phenotype, indicating that Nap positively regulates the formation of biofilm in A. brasilense Sp245.