Objectives: An ambulance can be a potential source of contagious or droplet infection of a community. We estimated the prevalence of positive carriage of tuberculosis (TB), methicillin-resistant $Staphylococcus$ $aureus$ (MRSA), and vancomycin-resistant $Enterococci$ (VRE) in patients transported by ambulance. Methods: This was a retrospective observational study. We enrolled all patients who visited a tertiary teaching hospital emergency department (ED). Blood, sputum, urine, body fluid, and rectal swab samples were taken from patients when they were suspected of TB, MRSA, or VRE in the ED. The patients were categorized into three groups: pre-hospital ambulance (PA) group; inter-facility ambulance (IA) group; and non-ambulance (NA) group. Adjusted odds ratio (OR) and 95% confidence intervals (CI) were calculated using a multivariable logistic regression model for the prevalence of each infection. Results: The total number of patients was 89206. Of these, 9378 (10.5%) and 4799 (5.4%) were in the PA and IA group, respectively. The prevalence of TB, MRSA, and VRE infection were 0.3%, 1.1%, and 0.3%, respectively. In the PA group, the prevalence of TB, MRSA, and VRE were 0.3%, 1.8%, and 0.4%. In the IA group, the prevalence of TB, MRSA, and VRE were 0.7%, 4.6%, and 1.5%, respectively. The adjusted ORs (95% CI) of the PA and IA compared to the NA group were 1.02 (0.69 to 1.53) and 1.83 (1.24 to 2.71) for TB, 2.24 (1.87 to 2.69) and 5.47 (4.63 to 6.46) for MRSA, 2.59 (1.78 to 3.77) and 8.90 (6.52 to 12.14) for VRE, respectively. Conclusions: A high prevalence of positive carriage of TB, MRSA, and VRE in patients transported by metropolitan ambulances was found.