International literature data report that the increase of infectious risk may be due to heating, ventilation and air conditioning (HVAC) systems contaminated by airborne pathogens. Moreover, the presence of complex rotating dehumidification wheels (RDWs) may complicate the cleaning and disinfection procedures of the HVAC systems. We evaluated the efficacy of a disinfection strategy applied to the RDW of two hospitals’ HVAC systems. Hospitals have four RDW systems related to the surgical areas (SA1 and SA2) and to the intensive and sub-intensive care (IC and sIC) units. Microbiological air and surface analyses were performed in HVAC systems, before and after the disinfection treatment. Hydrogen peroxide (12%) with silver ions (10 mg/L) was aerosolized in all the air sampling points, located close to the RDW device. After the air disinfection procedure, reductions of total microbial counts at 22 °C and molds were achieved in SA2 and IC HVAC systems. An Aspergillus fumigatus contamination (6 CFU/500 L), detected in one air sample collected in the IC HVAC system, was eradicated after the disinfection. The surface samples proved to be of good microbiological quality. The results suggest the need for a disinfection procedure to improve the microbiological quality of the complex HVAC systems, mostly in surgical and intensive care areas.