Selected photochemical and photophysical parameters of flavin mononucleotide (FMN) have been examined under conditions in which FMN is (1) solvated in a buffered aqueous solution, and (2) encased in a protein likewise solvated in a buffered aqueous solution. The latter was achieved using the so-called “mini Singlet Oxygen Generator” (miniSOG), an FMN-containing flavoprotein engineered from Arabidopsis thaliana phototropin 2. Although FMN is a reasonably good singlet oxygen photosensitizer in bulk water (ϕΔ = 0.65 ± 0.04), enclosing FMN in this protein facilitates photoinitiated electron-transfer reactions (Type-I chemistry) at the expense of photosensitized singlet oxygen production (Type-II chemistry) and results in a comparatively poor yield of singlet oxygen (ϕΔ = 0.030 ± 0.002). This observation on the effect of the local environment surrounding FMN is supported by a host of spectroscopic and chemical trapping experiments. The results of this study not only elucidate the behavior of miniSOG but also provide useful information for the further development of well-characterized chromophores suitable for use as intracellular sensitizers in mechanistic studies of reactive oxygen species.