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This research aims to evaluate the relationship between the environmental and design parameters of the air conditioning system, and the presence of colony-forming units (CFUs) of bacteria on surfaces. This retrospective study was carried out in controlled environment rooms in 12 hospitals in Spain, as part of a periodic environmental biosecurity assessment programme. Between 2010 and 2017, a total amount of 1027 samples were collected from floors and 861 from hand contact surfaces (HCSs), and the environmental parameters (temperature, relative humidity, fungal and bacterial CFUs present in the air) and design parameters (air changes per hour (ACH), differential pressure and position of the high efficiency particulate air filter) were monitored. The multivariate analysis concluded that, for each increased degree Celsius of temperature, the bacterial CFUs increased by 22.4% (incidence rate ratio (IRR): 1.224, 1.05–1.43) and by 25.9% (IRR: 1.259, 1.06–1.50) on HCSs and floors, respectively, and for each increase in ACH, the CFUs decreased 3.9% (IRR: 0.961, 0.94–0.99) and 2.5% (IRR: 0.975, 0.95–1.00) on HCSs and floors, respectively. Our results show the influence of the environmental and design parameters of air conditioning on the number of CFUs on surfaces and floors