Aim: Determine the utility of the Periodic Environmental Biosecurity Assessment Program (PEBAP) in achieving clean air as measured by the number of colony-forming units (CFU) of fungi and bacteria in the air. Background: There is no international consensus on the sampling frequency, the recommended limits for microorganisms in the air nor on the usefulness of routine microbiological air monitoring of hospitals. Methods: During the PEBAP, data were recollected between 2010 and 2017 in eight hospitals in southeast Spain. Air samples were collected in very high risk rooms (VHRRs) and high risk rooms (HRRs), unoccupied, using active sampling methods. Temperature, relative humidity, air changes per hour (ACH), and differential pressure were measured. When limits of CFU of opportunistic fungi and bacteria established in the PEBAP were exceeded, corrective measures were adopted. Results: We found a reduction ( p < .01) of percentage of air samples with fungi growth throughout the years of PEBAP in all rooms. Aspergillus was the most frequent opportunistic fungus. We found a high compliance of the standards of CFU of bacteria in HRR, and the percentage of compliance in VHRR was lower than in HRR in all years. Differences in environmental and design parameters were statistically significant ( p < .05) between rooms, except for ACH. Conclusions: PEBAP resulted in a useful tool to maintain and improve air quality in hospitals. The control of environmental biosecurity requires a multidisciplinary approach from preventive medicine, engineering, and cleaning services. Aspergillus is the most frequent opportunistic fungus in southeast Spain.