We demonstrate the applicability of near-infrared (NIR) autofluorescence (AF) of skin tissues to differentiating neoplasms based on performing a series of experiments with in vivo and ex vivo skin tumors and analyzing the skin AF spectral shape, the excitation–emission matrices, and the photobleaching properties of malignant and benign neoplasms. The melanin-pigmented lesions showed an increase of the AF in comparison to nonpigmented tissue fluorescent emission using excitation at 785 nm. Autofluorescent spectral differences can be associated with different concentration of melanocytes cells in the investigated skin lesions. The differences in excitation–emission matrices for tested tissues prove that melanin is the dominant NIR fluorophore in human skin. Further, we found that the photobleaching properties of normal skin and neoplasms differ significantly. The highlighted differences in skin tissue AF response can be used in rapid analysis of large tissue areas and can complement other methods of skin tumor detection.