Ultraviolet light-emitting diode (UV-LED) is an emerging UV source with many special features due to the nature of semiconductor devices. One such feature is wavelength diversity that does not exist in conventional mercury based UV lamps, which provides opportunities to selectively combine multiple wavelengths for potentially additional effects by UV-LEDs. In this work, the inactivation of different microorganisms in water was investigated by UV-LEDs wavelength combinations. Various wavelength combinations, including simultaneous and sequential exposures, in different UV ranges such as UVC, UVB and UVA, were examined. These combinations were applied to the inactivation of indicator bacterium E. coli and coliphage MS2 in water. The results showed the effect of UV-LEDs multiple wavelengths depends on which wavelengths (UVC, UVB and UVA) are combined and the manner that different wavelengths (simultaneous, sequential) are used. Also, different microorganisms (bacteria, virus) respond differently to wavelength combinations. Combinations of UVC/UVB always achieved additive effect on microorganisms inactivation due to the same photochemical reaction induced by UVC/UVB on DNA. Combining UVA with UVC/UVB simultaneously or applying UVA after UVC/UVB reduced the inactivation of bacterium E. coli due to DNA repair and photoreactivation effect of UVA. However, applying extended UVA exposure before UVC significantly improved E. coli inactivation. For virus MS2 inactivation, only additive effect was observed under various wavelength combinations. This study presented a comprehensive work on UV-LEDs wavelength combinations, which is of significance on the application of UV-LEDs for water disinfection. Display omitted •UV-LEDs wavelength combinations were thoroughly investigated for water disinfection.•Inactivation of different types of microorganisms, E. coli and MS2, was examined.•The effect of multiple wavelengths depends on UV ranges and combining manners.•UVC- and UVB-LEDs combinations always achieved additive inactivation effect.•UVA may improve or reduce E. coli inactivation depending on the manner to apply UVA.