DNA-stabilized silver nanoclusters (DNA-AgNCs) are a class of fluorophores with interesting photophysical properties. They are capable of generating anti-Stokes fluorescence upon excitation with near-infrared lasers. The anti-Stokes fluorescence has previously been speculated to be either the result of consecutive photon absorption (upconversion) or hot band absorption (HBA). Here, we revisit the anti-Stokes fluorescence of DNA-AgNCs to determine the underlying mechanistic origin. We investigate two previously studied DNA-AgNCs together with two organic fluorophores with absorption and emission features in the same spectral regions as the DNA-AgNCs. From the recorded anti-Stokes fluorescence excitation spectra, we find that all the emitters exhibit an exponential-like decaying slope on the red side of the lowest energy absorption band. Furthermore, excitation power dependency measurements at different excitation wavelengths verify the one-photon nature of the anti-Stokes fluorescence. Ultimately, the lack of discrete optical transition features in the anti-Stokes fluorescence excitation spectra suggests that the HBA mechanism is the most plausible cause for the anti-Stokes fluorescence of the investigated DNA-AgNCs.