Viral aerosols can lead to respiratory viral infections with high infectivity. About 90% of people’s time is spent in closed environments. A few studies have pointed out that the ventilation systems in air handling units (AHUs) that treat and transmit a new synthetic clean and conditioned environment can also spread and transport viral particles in buildings. The aim of this work is to study the characterization of adenovirus, a DNA non-enveloped respiratory virus, on the F7 fiberglass filter used in AHUs. In this study, an experimental setup simulating an AHU was used. The SYBR® QPCR, Electrical Low-Pressure Impactor (ELPI™) and Scanning Mobility Particle Sizer (SMPS™) were used to detect, measure and characterize the aerosolized adenovirus solution. The characterization results showed that the nebulized adenovirus could be aerosolized in different forms associated or not with cell debris and proteins. The quantification and level of infectivity of adenovirus demonstrated that viruses passed through filters and remained infectious up- and downstream of the system during the 25 min of aerosolization. This study showed that AHUs should be considered an indoor source of viral contamination. © 2019, Springer Nature B.V.