Several yeast species are able to synthesize and accumulate UV-radiation-absorbing mycosporine metabolites that are of unclear physiological function. In this work we analyzed the relationship between mycosporine-glutaminol glucoside (MGG) production, cell survival after UVB irradiation, and formation of cyclobutane pyrimidine dimers (CPDs). We also assessed the photostability and singlet oxygen quenching activity of MGG. A set of nine isolates of the basidiomycetous yeast Cryptococcus steppossus cultured in both dark and light conditions was used for the studies. Survival of the UVB-irradiated isolates and MGG concentration had a linear relationship when the concentration was over 2.5 mg g−1. CPD accumulation and MGG accumulation were inversely related. MGG in aqueous solution was photostable with a photodecomposition quantum yield of 1.16 × 10−5. MGG quenching of singlet oxygen was also observed, and the rate constant for the process in D2O was 5.9 × 107 M−1 s−1. Our results support the idea that MGG plays an important role as a UVB photoprotective metabolite in yeasts by protecting against direct damage on DNA and probably against indirect damage by singlet oxygen quenching.