Ultra-violet C (UV-C) treatment is commonly used in sterilization processes in industry, laboratories, and hospitals, showing its efficacy against microorganisms such as bacteria, algae, or fungi. In this study, we have eradicated for the first time all proliferating biofilms present in a show cave (the La Glacière Cave, Chaux-lès-Passavant, France). Colorimetric measurements of irradiated biofilms were then monitored for 21 months. To understand the importance of exposition of algae to light just after UVradiation, similar tests were carried out in laboratory conditions. Since UV-C can be deleterious for biofilm support, especially parietal painting, we investigated their effects on prehistoric pigment. Results showed complete eradication of cave biofilms with no algae proliferation observed after 21 months. Moreover, quantum yield results showed a decrease directly after UV-C treatment, indicating inhibition of algae photosynthesis. Furthermore, no changes in pigment color nor in chemical and crystalline properties has been demonstrated. The present findings demonstrate that the UV-C method can be considered environmentally friendly and the best alternative to chemicals. This inexpensive and easily implemented method is advantageous for cave owners and managers.