Serpentoviruses (order Nidovirales) are an important cause of respiratory disease in snakes. Although transmission studies have shown that serpentoviruses can cause respiratory disease in pythons, the possible role of additional potential pathogens is not yet understood. Very little information is available on the role of mycoplasma and chlamydia infections in disease in pythons. Diagnostic samples from 271 pythons of different genera submitted to a laboratory for detection of serpentoviruses were also screened for mycoplasma and chlamydia infections by PCR. Most of the samples were oral swabs. Almost 30% of the samples were positive for serpentoviruses, and mycoplasmas were detected in more than 60% of the pythons. The occurrence of these two pathogens correlated significantly (P < 0.001). Additionally, about 3% of the snakes tested positive for Chlamydia. This study found a high prevalence of mycoplasmas in the tested pythons and a correlation between infections with these bacteria and serpentoviruses in python samples submitted for diagnostic testing. Because the role mycoplasmas play in respiratory diseases of snakes is still largely unknown, further investigations are necessary to evaluate the role of mixed infections in disease.