This study was conducted to evaluate the effect of experimental infection with Escherichia coli O2 and H9N2 influenza virus on inflammatory factors in broiler chickens. A total of 120 oneday-old Cobb broiler chicks were divided randomly to 6 groups. Inoculation program with 10 9 EID50/bird of the A/Chicken/Iran/772/1998 (H9N2) virus and 10 9 CFU/mL/bird of E. coli O2 was carried out as follows: the chicks in group 1 were inoculated with virus and bacteria simultaneously on day 26, group 2 received virus on day 26 and then bacteria 3 days later, group 3 were inoculated with bacteria on day 23 and then virus on day 26, group 4 received only bacteria on day 26, group 5 were inoculated with only virus on day 26 and group 6 served as control. Serum samples were collected from wing vein at days 20, 30, and 40. Sera were examined for inflammatory mediators (TNF-a and INF-γ), acute phase reactants (haptoglobin and serum amyloid A) and gangliosides (total, lipid-bound and protein-bound sialic acids) using validated standard procedures. Among the measured parameters, serum gangliosides showed significant differences between the challenged and control groups in different days post inoculation (P<0.05). Significant increase in serum concentrations of serum sialic acids was observed on the 30 th day in challenged groups. Elevations were found in the concentrations of serum gangliosides on day 40 compared to their first concentrations. The most obvious increase in serum sialic acids was observed in group 1 challenged with avian influenza virus and E. coli O2 simultaneously. Bacterial infected group showed more significant changes in comparison with viral infected one. These findings suggest that serum sialic acids may be a useful indicator of H9N2 avian influenza virus and avian pathogenic E. coli O2 co-infection.