Quantum dot based-thermometry, in combination with double beam confocal microscopy and infrared thermal imaging, has been used to investigate the heating efficiency of multi-walled carbon nanotubes (MWCNTs) under optical excitation within the first (808 nm) and second (1090 nm) biological windows as well as in the spectral region separating them (980 nm). It has been found that for the three excitation wavelengths the heating efficiency of MWCNTs (10 nm in diameter and 1.5 μm in length) is close to 50%. Despite this "flat" heating efficiency, we have found that the excitation wavelength is, indeed, critical during in vivo experiments due to the spectral dependence of both tissue absorption and scattering coefficients. It has been concluded that efficiency and selectivity of in vivo photothermal treatments based on MWCNTs are simultaneously optimized when laser irradiation lies within the first or second biological window.