Ambulance vehicles are an essential part of emergency clinical services. Bioburden control in ambulances, through cleaning and disinfection, is crucial to minimize hospital-acquired infections, cross contamination and exposure of patients and ambulances‘ crew. In Portugal, firefighter crews are responsible, besides fire extinction, for first aid and urgent pre-hospital treatment. This study assessed the bioburden in Portuguese firefighters’ ambulances with a multi-approach protocol using active and passive sampling methods. Fungal resistance profile and mycotoxins detection in ambulances' ambient, and S. aureus (SA) prevalence and resistance profile in ambulances' ambient and colonization in workers were also investigated. Toxigenic fungi with clinical relevance, namely Aspergillus section Fumigati, were found on ambulance's air in the hazardous dimension range. Interestingly, surface contamination was higher after cleaning in several sampling sites. Prevalence of S. aureus was 3% in environmental samples, of which 2% were methicillin-sensitive (MSSA) and 1% methicillin-resistant (MRSA). About 2.07 fungal species were able to grow in at least one azole, ranging from one (44% samples) to five (6% samples) species in each azole. Mycotoxins were detected in mops and electrostatic dust cloths. Colonization by S. aureus in the firefighter crew was observed with a high associated prevalence, namely 48%, with a 24% prevalence of MSSA (8/33) and 21% of MRSA (7/33). Additional studies are needed to determine the potential risk of infection transmission between different vehicle fleets and under varying conditions of use. This will strengthen the paramedic sector's mission to save lives without putting their own health and safety at risk.