Two chemically synthesized flavin derivatives, 8-trifluoromethyl- and 8-bromoriboflavin (8-CF3RF and 8-BrRF), were photochemically characterized in H2O and studied spectroscopically after incorporation into the LOV domain of the blue light photoreceptor YtvA from Bacillus subtilis. The spectroscopic studies were paralleled by high-level quantum chemical calculations. In solution, 8-BrRF showed a remarkably high triplet quantum yield (0.97, parent compound riboflavin, RF: 0.6) and a small fluorescence quantum yield (0.07, RF: 0.27). For 8-CF3RF, the triplet yield was 0.12, and the fluorescence quantum yield was 0.7. The high triplet yield of 8-BrRF is due to the bromine heavy atom effect causing a stronger spin–orbit coupling. Theoretical calculations reveal that the decreased triplet yield of 8-CF3RF is due to a smaller charge transfer and a less favorable energetic position of T2, required for intersystem crossing from S1 to T1, as an effect of the electron-withdrawing CF3 group. The reconstitution of the LOV domain with the new flavins resulted in the typical LOV photochemistry, consisting of triplet state formation and covalent binding of the chromophore, followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties.