Recently seven isolates of avian influenza virus (AIV) serotype H9N2 recovered from an outbreak of AI were analyzed on the basis of their biological and molecular characteristics. All the isolates belonged to the low-pathogenicity group of AIV. To further evaluate their pathogenic potential in association with other organisms, an isolate was inoculated experimentally in chickens using different routes and subsequently challenged with infectious bronchitis virus, Ornithobacterium rhinotracheale or Escherichia coli. The virus isolation and seromonitoring data revealed a significant role of Escherichia coli in aggravating the clinical condition of the birds earlier infected with AIV (H9N2). The AIV-antigen was detected in lung, trachea, kidney, and cloacal bursa among the infected birds, using immunofluorescent antibody technique. In another experiment, chickens that were immunosuppressed chemically showed high mortality when challenged with AIV H9N2. The results indicated that this low pathogenicity AIV (H9N2) isolate could produce severe infection depending on the type of secondary opportunistic pathogens present under field conditions. This may explain the severity of infection with the present H9N2 outbreak in the field. A prolonged antibacterial therapy in flocks infected with AIV H9N2 and use of oil-based vaccine at an early age in new flocks has helped to control this infection and the disease.