Aims This study was designed to investigate, in an in vivo setting, the effects of single and combined infections with either Mycoplasma gallisepticum (MG) and/or Escherichia coli on the chicken immune response induced by Newcastle disease virus (NDV) vaccine. Methods and Results Humoral immunity was measured through detection of NDV antibody and anti-NDV IgG titres using haemagglutination-inhibition test and enzyme-linked immunosorbent assay, respectively. In addition, the expression levels of pro-inflammatory cytokines' genes (interleukin (IL) 6, IL4 and interferon (IFN) gamma) were analysed using quantitative reverse transcription PCR. Significant (P < 0 center dot 05) results in all immunological parameters were detected in the vaccinated noninfected chicken group in comparison with those in groups exposed to bacterial infections. Bacterial infection along with vaccination hampered the NDV antibodies production and reduced the vaccine upregulated cytokine genes. The vaccinated mixed infection group reported lower antibody titres and cytokines expression levels compared to those in the single infection groups. All the previously enhanced immunological parameters reflected the maximum protection post challenge with velogenic viscerotropic NDV in the vaccinated noninfected chicken group. Conclusions These findings provide novel insights into the immunosuppression activities of MG and E. coli infection in chickens vaccinated against NDV. Significance and Impact of the Study This study hopes to provide a better insight to the immunosuppressive action of bacterial pathogens in chickens. This will help to improve biosecurity strategies during NDV vaccination in the future.