The combined use of fluorescence diagnostics (FD) and photodynamic therapy (PDT) is a promising approach to the treatment of cholangiocarcinoma. Information about the probing depth of laser radiation at a therapeutic dose sufficient for the appearance of the photodynamic effect allows planning the PDT process. We aim to assess the probing depth of radiation at a therapeutic dose. The highest fluorescence intensity is observed in the gall bladder and liver tissues. A significant difference was noted in the intensity of backscattered laser radiation, depending on the segments of the wild boar hepatobiliary system. Analysis of the probing depth of radiation (λ = 660 nm) revealed that when the optical fiber is located outside the drainage, it increases by 2 to 4 mm, which improves the efficacy of treatment. The applicability of video FD of segments of the hepatobiliary system is investigated using the two-channel video fluorescence system. During video FD in the near-infrared range, the contrast of the fluorescence images of the hepatobiliary system is less than in the visible range. The results of the study will improve the quality of diagnostic information and optimize the FD and PDT algorithms for malignant neoplasms of the human hepatobiliary system.