Recently, ultraviolet light-emitting diodes (UV-LEDs) have emerged as a new UV source, bringing flexibility for various UV wavelength combinations due to their unique feature of wavelength diversity. In this study, we investigated inactivation mechanisms of representative microorganisms at different wavelength combinations using UV-LEDs. Two types of indicator microorganisms were examined, namely Escherichia coli (E. coli) as a representative bacteria and bacteriophage MS2 as a representative virus. Different inactivation effects were observed, and the results for UVA pretreatment followed by UVC inactivation were particularly interesting. While a substantial shoulder in the E. coli UVC inactivation curve was observed, this was reduced by UVA pretreatment (365 nm) at 17 J/cm2. Further, 52 J/cm2 UVA eliminated the shoulder in the fluence-response curves, resulting in improved UVC (265 nm) inactivation of E. coli by over two orders of magnitude. No inactivation improvement was observed for MS2. Moreover, UVA pretreatment eliminated photoreactivation of E. coli but did not affect dark repair. Detailed investigation of inactivation mechanisms revealed that hydroxyl radicals (•OH) played a significant role in the effects of UVA pretreatment. This study demonstrated that •OH radicals were generated inside E. coli cells during UVA pretreatment, which accounted for the subsequent effects on E. coli. The impact of UVA pretreatment on E. coli inactivation and reactivation was mainly due to increased levels of •OH radicals in E. coli cells, impairing cell functions such as DNA self-repair. Display omitted •Inactivation mechanisms of UVA and UVC wavelength combinations were investigated.•UVA pretreatment largely improved E. coli inactivation and reduced reactivation.•Hydroxyl radicals were generated inside E. coli cells during UVA pretreatment.•Hydroxyl radicals played a significant role in the effects of UVA pretreatment.•E. coli cell function like DNA repair was impaired, causing improved inactivation.