Objectives: We sought to assess microbial air quality and bacterial surface contamination on medical instruments and the surrounding areas among 30 ambulance runs during service. Methods: We performed a cross-sectional study of 106 air samples collected from 30 ambulances before patient services and 212 air samples collected during patient services to assess the bacterial and fungal counts at the two time points. Additionally, 226 surface swab samples were collected from medical instrument surfaces and the surrounding areas before and after ambulance runs. Groups or genus of isolated bacteria and fungi were preliminarily identified by Gram's stain and lactophenol cotton blue. Data were analyzed using descriptive statistics, t-test, and Pearson's correlation coefficient with a p-value of less than 0.050 considered significant. Results: The mean and standard deviation of bacterial and fungal counts at the start of ambulance runs were 318±485cfu/m3 and 522±581cfu/m3 , respectively. Bacterial counts during patient services were 468±607cfu/m3 and fungal counts were 656±612cfu/m3 . Mean bacterial and fungal counts during patient services were significantly higher than those at the start of ambulance runs, p=0.005 and p=0.030, respectively. For surface contamination, the overall bacterial counts before and after patient services were 0.8±0.7cfu/cm2 and 1.3±1.1cfu/cm2 , respectively (p<0.001). The predominant isolated bacteria and fungi were Staphylococcus spp. and Aspergillus spp., respectively. Additionally, there was a significantly positive correlation between bacterial (r=0.3, p<0.010) and fungal counts (r=0.2, p=0.020) in air samples and bacterial counts on medical instruments and allocated areas. Conclusions: This study revealed high microbial contamination (bacterial and fungal) in ambulance air during services and higher bacterial contamination on medical instrument surfaces and allocated areas after ambulance services compared to the start of ambulance runs. Additionally, bacterial and fungal counts in ambulance air showed a significantly positive correlation with the bacterial surface contamination on medical instruments and allocated areas. Further studies should be conducted to determine the optimal intervention to reduce microbial contamination in the ambulance environment