Emergency medical service (EMS) providers face significant exposure to infectious aerosols during outbreaks like the COVID-19 pandemic. Most ambulances lack ventilation controls to reduce EMS worker exposure to these aerosols. Ambulances are smaller than hospital rooms and handle numerous patients daily, increasing contact with potentially infectious individuals. Ventilation controls such as portable high-efficiency particulate air (HEPA) filtration can mitigate this risk. Few studies have assessed portable HEPA filters in ambulances. This study evaluated two HEPA filter models in an unoccupied, stationary research ambulance at the National Institute for Occupational Safety and Health (NIOSH) in Cincinnati. A tracer aerosol simulated patient aerosol generation, and optical particle counters (OPCs) measured aerosols. The HEPA units were tested individually, placed in the same location, and operated for 50 min. Results showed significant reductions in aerosol concentrations during the generation phase, with performance varying during the decay period. Overall, HEPA units reduced particle concentrations by around 50% during the generation phase and continued to be effective through the decay period. This demonstrates the potential of portable HEPA filters as an affordable and effective option for air cleaning in ambulance patient modules.