Basal cell carcinoma (BCC) is the most common type of skin cancer. Diagnosis and edge assessment of BCC lesions are based on clinical and dermoscopy evaluation, which are strongly dependent on the expertise and training of the physician. There is a high rate of underdiagnosis because BCC is frequently confused with certain common benign lesions and is often indistinguishable from the surrounding healthy tissue. In the present study, a multispectral fluorescence lifetime imaging (FLIm) dermoscopy system, designed for imaging and analyzing the autofluorescence emission of skin tissue, was used to image thirty-eight patients with diagnosed nodular BCC (nBCC) lesions, using clinically acceptable levels of excitation light exposure. With this system, skin autofluorescence was imaged simultaneously using three emission bands: 390 ± 20 nm, 452 ± 22 nm, and >496 nm, preferentially targeting collagen, NADH, and FAD autofluorescence, respectively. Statistical classifiers based on FLIm features developed to discriminate BCC from healthy tissue showed promising performance (ROC area-under-the-curve of 0.82). This study demonstrates the feasibility of clinically performing multispectral endogenous FLIm dermoscopy providing baseline results indicating the potential of this technology as an image-guided tool to improve the delineation of nBCC during surgical lesion resection.