Improving and optimizing photosensitizers for photodynamic wound therapy remains an urgent task in this field. This study investigates the properties of an ethosomal chlorin e6 (Ce6) photosensitizer and its penetration into various tissues within a wound area. We obtained and analyzed spatial- and geometric configurations, absorption and fluorescence spectra of Ce6 in both molecular and ethosomal forms, alongside studying its penetration into skin and muscle tissue. The synthesized particles were spherical with convex surfaces. The average particle diameter in the samples was 47.7 nm. The absorption and fluorescence spectra of ethosomal Ce6 exhibited a bathochromic shift of 13–16 nm relative to molecular Ce6. This shift in the fluorescence peak was primarily attributed to the presence of lecithin in the ethosomal solution. In contrast, the variability in peak position along the wavelength axis (2–5 nm) was more likely due to ethanol in the solution. Encapsulation of Ce6 in ethosomes overcomes the stratum corneum barrier of the skin, providing 1.5- to 2-fold deeper penetration (up to 180 μm) into the epidermis and papillary dermis compared to free Ce6 (50–120 μm). Upon direct application to muscle tissue, which lacks an epithelial barrier, both formulations penetrate substantially deeper (up to 1500 μm for ethosomal Ce6); however, tissue heterogeneity introduces considerable variability. Therefore, when planning diagnosis or therapy using ethosomal Ce6, it is necessary to select irradiation sources with wavelengths corresponding to these new, shifted absorption peaks, along with appropriate filters for detecting fluorescence in the new spectral range. Furthermore, the potentially greater and more variable depth of tissue saturation compared to skin must be taken into account, as this is a decisive factor in determining the optimal parameters for laser or light-emitting diode irradiation.