Avian influenza and infectious laryngeotracheitis viruses are common causes of respiratory diseases in chickens with economical importance worldwide. In this study, we investigated the effect of experimental co-infection of avian influenza virus-H9N2 (AIV-H9N2) with infectious laryngeotracheitis virus (ILTV) live-attenuated vaccine (LAR-VAC?) on chickens. Four experimental groups were included in this study: negative control group, AIV-H9N2 group, AIV-H9N2+LAR-VAC? group, and LAR-VAC? group. AIV-H9N2 was inoculated intranasally to challenged groups at 35 days of age. On the same day, LAR-VAC? was ocularly administered to vaccinated groups. Chickens were observed for clinical signs, changes in body weight and mortality rates. Tissue samples, sera, tracheal and cloacal swabs, and blood were also collected at 3, 6, 9 and 12 days post-infection (PI). A significant increase in clinical signs and mortality rates were observed in the AIV-H9N2?+?LAR-VAC? group. Moreover, chickens coinfected with AIV-H9N2 and LAR-VAC? showed a significant decrease in body weight and lymphoid organs indices. The tracheal gross and histopathological lesions and the shedding titer and period of AIV-H9N2 were significantly higher in AIV-H9N2?+?LAR-VAC? group when compared to other groups. Furthermore, AIV-H9N2 infection leads to humoral and cellular immunosuppression as shown by a significant decrease in the CD4+/CD8+ ratio and antibody responses to ILTV and a significant increase in H/L ratio. In conclusion, this is the first report of co-infection of AIV-H9N2 and ILTV vaccine in chickens, which leads to increased pathogenicity, pathological lesions, and AIV-H9N2 shedding titer and period, which can lead to severe economic losses due to poor weight gain and mortality.