Starlings have tremendous economic and environmental impacts because they can spread pathogens to livestock and poultry. These birds act as mechanical and biological carriers for different types of pathogens from and to their original habitat. The goal of this study was to ascertain the presence of Mycoplasma gallisepticum (MG) in the starlings’ lungs and confirm their diagnosis using the PCR technique. We altered the supplements that were added to Mycoplasma culture media by using calf serum instead of horse serum and sulphadimidine plus trimethoprim with nystatin instead of thallous acetate. Eighty-five starlings were bought from hunters in the spring of 2019, and their lungs were harvested and divided into two portions, one for Mycoplasma cultivation and the other for DNA extraction. Fifty-nine (69.4%) samples were positive for Mycoplasma colonies, thereby yielding accurate results using alternative supplements in the culture media. PCR revealed the presence of Mycoplasma in 78.8% lung samples, while MG was detected in only 43.3% of the positive samples, indicating the presence of other species of Mycoplasma too. The current study is the first of its kind not only in Iraq but also in the world, investigating the presence of MG in the lungs of starling birds. This study revealed that MG is significantly prevalent in starlings and also suggests that other Mycoplasma species may be present in starlings.