Knowledge of the molecular effects of UV radiation (UVR) on bacteria can contribute to a better understanding of the environmental consequences of enhanced UV levels associated with global climate changes and will help to optimize UV-based disinfection strategies. In the present work, the effects of exposure to UVR in different spectral regions (UVC, 100-280 nm; UVB, 280-320 nm; and UVA, 320-400 nm) on the lipids and proteins of two bacterial strains (Acinetobacter sp. strain PT5I1.2G and Pseudomonas sp. strain NT5I1.2B) with distinct UV sensitivities were studied by mid-infrared spectroscopy. Exposure to UVR caused an increase in methyl groups associated with lipids, lipid oxidation, and also led to alterations in lipid composition, which were confirmed by gas chromatography. Additionally, mid-infrared spectroscopy revealed the effects of UVR on protein conformation and protein composition, which were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), oxidative damage to amino acids, and changes in the propionylation, glycosylation and/or phosphorylation status of cell proteins. Differences in the targets of UVR in the two strains tested were identified and may explain their discrepant UV sensitivities. The significance of the results is discussed from an ecological standpoint and with respect to potential improvements in UV-based disinfection technologies.