Ambulances may be a source of multiresistant microorganisms as patients' microbiotas may colonize healthcare personnel and ambulance's environment. Microbial growth was observed in 73% of ambulances samples: Inner passenger door's handle, steering wheel and left handle of the stretcher had a higher bacterial load. Coagulase-negative staphylococci were the most frequently isolated microorganism while methicillin susceptible S. aureus (MSSA) was only isolated in two of the ten sampled ambulances. Presence of clinically relevant bacterial contamination at particular BLSA locations suggests that disinfection of the studied BLSAs was not optimal and represents a potential risk of infection for the patients transferred to them. Adherence to infection control programs should be enhanced to increase the awareness and education of Emergency system responders and therefore safer patient care practices.