We aimed to know the risk-stratification-based prevalence of bacterial contamination of ambulance vehicle surfaces, equipment, and materials. This study was performed in a metropolitan area with fire-based single-tiered Basic Life Support ambulances. Total 13 out of 117 ambulances (11.1%) were sampled and 33 sites per each ambulance were sampled using a soft rayon swab and aseptic containers. These samples were then plated onto a screening media of blood agar and MacConkey agar. Specific identification with antibiotic susceptibility was performed. We categorized sampling sites into risk stratification-based groups (Critical, Semi-critical, and Non-critical equipment) related to the likelihood of direct contact with patients' mucosa. Total 214 of 429 samples showed positive results (49.9%) for any bacteria. Four of these were pathogenic (0.9%) (MRSA, MRCoNS, and K. pneumoniae), and 210 of these were environmental flora (49.0%). However, the prevalence (positive/number of sample) of bacterial contamination in critical, semi-critical airway, semi-critical breathing apparatus group was as high as 15.4% (4/26), 30.7% (16/52), and 46.2% (48/104), respectively. Despite current formal guidelines, critical and semi-critical equipments were contaminated with pathogens and normal flora. This study suggests the need for strict infection control and prevention for ambulance services.