Objective: evaluate the cleaning and disinfection procedures (CDP) in six ambulances from three different entities in Pereira (Risaralda-Colombia). Methods: cross-sectional descriptive study frequencies of presence/absence were calculated from data obtained in the bacterial growth results before and after CDP, taking samples in three different places on the ambulances, being: back door, stretcher and wall next to the patient, before and after said processes. Additionally, surveys were carried out at the domicile of companies providing pre-hospital transport service. Results: 77.8% of the samples were positive. The most frequent morphology in the study was gram-positive cocci, which remained in a greater proportion after disinfection. At the microbiologic level, most of these suggested being Staphylococcus aureus-type. By implementing CDP, microbiological isolates were eliminated in 33.3%, being the door the ambulance area, which showed the greatest decrease (50.0%). Conclusions: A high prevalence of potentially pathogenic microorganisms exists in non-critical points of emergency ambulances. With CDPs, a decrease in microorganisms is achieved, but not their elimination, leaving in evidence that different factors must be considered in order to improve these CDP.