Objectives: Assessment of microbial air quality and surface contamination in ambulances and administration offices as a control place without occupational exposure to biological agents; based on quantitative and qualitative analysis of bacteria, yeasts and filamentous fungi found in collected samples. Material and Methods: The sampling was done by wet cyclone technology using the Coriolis recon apparatus, imprint and swab methods, respectively. In total, 280 samples from 28 ambulances and 10 offices in Warszawa were tested. Data was analyzed using Shapiro-Wilk normality test, Kruskal-Wallis test with α = 0.05. P value ≤ 0.05 was considered as significant. Results: The levels of air contamination were from 0 to 2.3×10¹ colony-forming unit (CFU)/m³ for bacteria and for yeast and filamentous fungi were from 0 to 1.8×10¹ CFU/m³. The assessment of office space air samples has shown the following numbers of microorganisms: bacteria from 3.0×10¹ to 4.2×10¹ CFU/m³ and yeast and filamentous fungi from 0 to 1.9×10¹ CFU/m³. For surface contamination the mean bacterial count in ambulances has been between 1.0×10¹ and 1.3×10² CFU/25 cm² and in offices – between 1.1×10¹ and 8.5×10¹ CFU/25 cm². Mean fungal count has reached the level from 2.8×10⁰ to 4.2×10¹ CFU/25 cm² in ambulances and 1.3×10¹ to 5.8×10¹ CFU/25 cm² in offices. The qualitative analysis has revealed the presence of Acinetobacter spp. (surfaces), coagulase – negative Staphylococci (air and surfaces), Aspergillus and Penicillium genera (air and surfaces). Conclusions: The study has revealed a satisfactory microbiological quantity of analyzed air and surface samples in both study and control environments. However, the presence of potentially pathogenic microorganisms in the air and on surfaces in ambulances may endanger the medical emergency staff and patients with infection. Disinfection and cleaning techniques therefore should be constantly developed and implemented. Int J Occup Med Environ Health 2017;30(4):617–627