Respiratory diseases are common in horses; however, there is a lack of information in the literature on respiratory disease affecting mule foals. The objective of this study was to investigate the presence of aerobic bacteria in tracheal wash samples from 20 mule foals up to 6 months of age, with and without clinical evidence of respiratory disease. Samples were collected via endoscopy in two separate occasions and sent for cytology, microbial culture, and PCR for detection of Rhodococcus equi. Based on clinical evidence, 32.5% (13/40) of the samples were obtained from mule foals displaying signs of a respiratory condition, whereas 50% (20/40) of the samples showed cytologic evidence of respiratory tract infection. One hundred percent of samples provided positive cultures with Escherichia coli (45%), Enterococcus (37.5%), and coagulase-negative Staphylococcus (30%) being the most common bacterial genera isolated. R. equi was not identified in any of the samples. The correlation between isolated bacterial agents and the presence of respiratory infection was not statistically significant. The microorganisms found in the samples may be naturally present in the soil, feces, and environment in which the animals live, presenting a risk of opportunistic respiratory infection.