Algal biofilms in caves visited by tourists are a significant threat to karstic formations. They cause a dirty greenish appearance on cave formations and provoke biophysical and biochemical erosion. Seeking an alternative to the use of chemicals, we investigated the effects of high doses of UV-C irradiation on the green algae Chlorella minutissima-dominating biofilms in the Moidons Cave (Jura, France). The algae were harvested and subjected to laboratory subculturing under conditions similar to the conditions in the cave (low temperatures and a short photoperiod with weak light intensities). After one or two doses of UV-C of 150 or 300 kJ m−2, the samples were incubated for 21 days, and the physiological parameters (pigment concentrations and photosynthetic activity) were monitored every seven days. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were also measured to detect oxidative stress, and analyses of DNA integrity were performed. The results showed that all UV-C treatments caused chlorophyll bleaching with completely inhibited metabolic activity. In addition, high UV-C doses caused oxidative stress and programmed cell death. Irradiation was tested on a natural biofilm that proliferated in the Moidons Cave. Colorimetric measurements of the treated biofilm indicated that UV-C treatment caused significant chlorophyll bleaching.