Infectious coryza (IC) is a respiratory disease in poultry caused by Avibacterium paragallinarum (Av. paragallinarum). The disease caused growth retardation in broilers and reduced egg production in laying hens, resulting in significant economic losses to the global chicken industry. The biofilm is an important virulence factor for many bacterial pathogens, yet there is a paucity of research on the biofilm of Av. paragallinarum. This study aimed to construct a random mutant library of Av. paragallinarum using the Tn5-Kan transposon to identify genes involved in biofilm formation. A total of approximately 3000 mutants were obtained, and 38 of them demonstrated a reduction in biofilm formation of 70-90 % by crystal violet staining. The transposon insertion sites were further determined by chromosome walking, and 17 functional genes related to biofilm formation were identified. According to the functional analysis of the mutated genes, 14 mutants with mutated genes associated with energy metabolism, cell membrane formation, gene transcription and translation, and material transmembrane transport were screened to further explore their biological characteristics and pathogenicity in vivo and in vitro. The results indicated that the growth performance, resistance to disinfectants, adhesion and invasion ability to DF-1 cells and pathogenicity of the 14 mutants were reduced. The 14 mutants displayed increased sensitivity to antibiotics but did not show significant changes in hemagglutination titer or antiserum bactericidal ability. It is noteworthy that the M-76 mutant exhibited a marked reduction in pathogenicity. Following challenge, the experimental chickens did not present any clinical symptoms or pathological changes for a period of seven days, and the respiratory tract bacterial shedding was also the lowest. This indicates that a deficiency in biofilm formation reduces the pathogenicity of Av. paragallinarum. This study will contribute to our understanding of the molecular mechanism of biofilm formation of Av. paragallinarum and further study the pathogenesis of Av. paragallinarum.