Mycoplasma infection is still very common in chicken and turkey flocks. Several low-pathogenicity avian influenza (LPAI) viruses are circulating in wild birds that can be easily transmitted to poultry flocks. However, the effect of LPAI on mycoplasma infection is not well understood. The aim of the present study was to investigate the infection of LPAI virus H3N8 (A/mallard/Hungary/19616/07) in chickens challenged with Mycoplasma gallisepticum. Two groups of chickens were aerosol challenged with M. gallisepticum. Later one of these groups and one mycoplasma-free group were aerosol challenged with the LPAI H3N8 virus. The birds were observed for clinical signs for 8 days, then euthanized, and examined for the presence of M. gallisepticum in the trachea, lung, air sac, liver, spleen, kidney and heart, and for developing anti-mycoplasma and anti-viral antibodies. The LPAI H3N8 virus did not cause any clinical signs but M. gallisepticum infection caused clinical signs, reduction of body weight gain and colonization of the inner organs. These parameters were more severe in the birds co-infected with M. gallisepticum and LPAI H3N8 virus than in the group challenged with M. gallisepticum alone. In addition, in the birds infected with both M. gallisepticum and LPAI H3N8 virus, the anti-mycoplasma antibody response was reduced significantly when compared with the group challenged with M. gallisepticum alone. Co-infection with LPAI H3N8 virus thus enhanced pathogenesis of M. gallisepticum infection significantly.