We assessed the disinfection efficacy of an ozone generator prototype in ambulances used to transport patients with coronavirus disease (COVID-19). This research consisted of three stages: in vitro tests using microbial indicators, such asCandida albicans, Escherichia coli, Staphylococcus aureusandSalmonella phage, which were experimentally inoculated onto polystyrene crystal surfaces within a 23 m3enclosure. They were then exposed to ozone at a 25 ppm concentration using the ozone generator (Tecnofood SAC) portable prototype, and the decimal reduction time (D) was estimated for each indicator. The second stage involved the experimental inoculation of the same microbial indicators on a variety of surfaces inside conventional ambulances. The third stage consisted of exploratory field testing in ambulances used to transport patients with suspected COVID-19. During the second and third stages, samples were collected by swabbing different surfaces before and after 25 ppm ozonisation for 30 min. Results suggested that ozone was most effective onCandida albicans(D = 2.65 min), followed byEscherichia coli(D = 3.14 min),Salmonella phage(D = 5.01 min) andStaphylococcus aureus(D = 5.40 min). Up to 5% of the microbes survived following ozonisation of conventional ambulances. Of the 126 surface samples collected from ambulances transporting patients with COVID-19, 7 were positive (5.6%) for SARS-related coronavirus as determined on reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Ozone exposure from the ozone generator prototype inside ambulances at a concentration of 25 ppm for 30 min can eliminate gram positive and negative bacteria, yeasts, and viruses.