Polychromatic ultraviolet (UV) light with bandwidth of 20 nm and peak emission centered at 224, 254, or 280 nm (UV224, UV254, and UV280, respectively) were used to inactivate human adenovirus type 2 (HAdV-2). Quantitative polymerase chain reaction (qPCR) and reverse transcriptase qPCR assays were used to elucidate the step in the HAdV-2 replication cycle that was disrupted after UV exposure. UV treatment at any of the wavelengths analyzed did not inhibit association of HAdV-2 to the host cells even after exposure to a fluence (UV dose) that would produce a virus inactivation efficiency, measured by plaque assay, greater than 99.99%. In contrast, UV irradiation at all three peak emissions disrupted early E1A gene transcription and viral DNA replication, but different mechanisms appeared to be dominating such disruptions. UV224 seemed to have little effect on the integrity of the viral genome but produced a structural transformation of the viral capsid that may inhibit the delivery of viral genome into the host cell nucleus. On the other hand, UV254 and UV280 did not affect the integrity of the viral capsid, but the mutations they produced on the viral genome might cause the inhibition of the early gene transcription and DNA replication after the viral genome successfully translocated into the host cell nucleus.