Avian influenza virus (AIV) poses significant threats to poultry and human health. This study investigates the impact of H9N2 AIV infection on the respiratory microbiota of chickens using 16S rRNA gene sequencing. Forty-eight one-day-old specific pathogen-free (SPF) chickens were assigned to six groups: a control and five post-infection groups (days 1, 3, 5, 7, and 9). After a 15-day microbiota stabilization period, the infected chickens received a viral inoculum (10^7 TCID50/mL) via ocular, intra-nasal, and intra-tracheal routes. Tracheal and broncho-alveolar lavage samples were analyzed. Significant reductions in microbiota diversity were observed on days 5, 7, and 9 post-infection, compared to d0 controls. PERMANOVA confirmed significant beta diversity differences (P = 0.001) between infected and uninfected groups. The microbial shifts from d5 to d9 were marked by increased Proteobacteria, decreased Actinobacteria and Firmicutes, and a rise in Dickeya. Elevated type I interferon (IFN-β) and viperin gene expression at d5 coincided with reduced microbiota diversity, highlighting the respiratory microbiota´s role in modulating host responses to AIV H9N2 infection and suggesting potential biomarkers for respiratory dysbiosis.