In skin cancer surgery, the rapid and accurate detection of surgical margin is required for precise and complete removal. Reflectance confocal microscopy (RCM) is currently used to guide the surgical margin, but has a limited detection contrast. In this study, we used a moxifloxacin based two-photon microscopy (moxi-TPM) as a high-speed and high-contrast method of human skin cancer. High-speed TPM was realized by using moxifloxacin as a cell-labeling agent. Various skin cancer tissues including squamous cell carcinoma, basal cell carcinoma, and highly pigmented melanoma were visualized in cellular level resolution by using moxi-TPM, and these moxi-TPM images were compared with dermoscopic images and histology. These results showed that moxi-TPM could be used for guiding skin cancer surgery owing to its high imaging speed and resolution.2