Adenovirus is regarded as the most resistant pathogen to ultraviolet (UV) disinfection due to its demonstrated resistance to monochromatic, low-pressure (LP) UV irradiation at 254 nm. This resistance has resulted in high UV dose requirements for all viruses in regulations set by the United States Environmental Protection Agency. Polychromatic, medium-pressure (MP) UV irradiation has been shown to be much more effective than 254 nm, although the mechanisms of polychromatic UV inactivation are not completely understood. This research analyzes the wavelength-specific effects of UV light on adenovirus type 2 by analyzing in parallel the reduction in viral infectivity and damage to the viral genome. A tunable laser from the National Institute of Standards and Technology was used to isolate single UV wavelengths. Cell culture infectivity and PCR were employed to quantify the adenoviral inactivation rates using narrow bands of irradiation (<1 nm) at 10 nm intervals between 210 and 290 nm. The inactivation rate corresponding to adenoviral genome damage matched the inactivation rate of adenovirus infectivity at 253.7 nm, 270 nm, 280 nm, and 290 nm, suggesting that damage to the viral DNA was primarily responsible for loss of infectivity at those wavelengths. At 260 nm, more damage to the nucleic acid was observed than reduction in viral infectivity. At 240 nm and below, the reduction of viral infectivity was significantly greater than the reduction of DNA amplification, suggesting that UV damage to a viral component other than DNA contributed to the loss of infectivity at those wavelengths. Inactivation rates were used to develop a detailed spectral sensitivity or action spectrum of adenovirus 2. This research has significant implications for the water treatment industry with regard to polychromatic inactivation of viruses and the development of novel wavelength-specific UV disinfection technologies.