Mycoplasma gallinaceum is not among the most pathogenic mycoplasmas affecting poultry, but its continuous re-isolation from flocks in South Africa displaying typical signs of mycoplasmosis prompted us to revisit its role in respiratory disease. Specific-pathogen-free white leghorn chickens were co-challenged with either M. gallinaceum (MGC) and QX-like infectious bronchitis virus (IBV), or the more virulent Mycoplasm gallisepticum (MG) and IBV. No clinical signs were observed apart from sneezing in chickens challenged with IBV, MGC IBV, and MG IBV. On postmortem examination, one bird each in the MGC IBV and IBV groups developed peritonitis or airsacculitis, respectively. In the tracheas, the MG IBV group showed the most severe ciliary damage with a mean ciliostatic score of 32.40 compared to scores of 26.83 and 20.4 for the MGC IBV and IBV groups, respectively. Corresponding tracheal lesions were recorded. Quantitation of the challenge pathogens by quantitative real-time PCR and real-time reverse transcriptase–PCR determined that MGC was shed in much higher titers from the trachea than MG, when co-infected with IBV. Interestingly, the presence of both MG and MGC appeared to enhance IBV replication in the tracheas of infected chickens, whereas the presence of IBV suppressed MG and MGC proliferation in the trachea. In general, the nonpathogenicity of M. gallinaceum in chickens was confirmed, but it was able to aggravate respiratory disease and pathogen proliferation with virulent QX-like IBV.