The ground and excited state (in the singlet state, S1) acid–base equilibria, together with the photophysical properties of the two main constituents of brazilwood, brazilin and brazilein, have been investigated in aqueous solutions in the pH range: −1 < pH < 10. Brazilin is the colorless reduced form of brazilein where three ground and three excited state species (B(red)H(n), with n = 2–4 representing the protonated hydroxyl groups) are observed with two corresponding acidity constants: pKa1 = 6.6 and pKa2 = 9.4 (pKa1* = 4.7 and pKa2* = 9.9, obtained from the Förster cycle). In the case of brazilein, three ground species (pKa1 = 6.5 and pKa2 = 9.5) and four excited state species were identified (again from the Förster cycle: pKa1* = 3.9 and pKa2* = 9.8). The colorless species (brazilin) presents a high fluorescence quantum yield (F = 0.33) and competitive radiative channel (kF = 1.3 × 10(9) s(–1)) over radiationless processes (kNR = 2.6 × 10(9) s(–1)). In contrast to this behavior, brazilein displays a F value 2 orders of magnitude lower and a dominance of the radiationless decay pathways, which is suggested to be linked to an excited state proton transfer leading to a quinoidal-like structure. This is further supported by time-resolved data (obtained in a ps time domain). The overall data indicates that brazilin is more prone to degradation than brazilein, mainly due to the high efficiency of the radiationless decay channel (likely through internal conversion), which confers a stabilizing inherent characteristic to the latter. In the case of brazilein, the efficiency of the radiationless channel is linked to an excited state intramolecular proton transfer resulting from an excited state equilibrium involving neutral and zwitterionic tautomeric species of this compound. Furthermore, a theoretical study has been performed with the determination of the optimized ground-state and excited molecular geometries for the two compounds together with the prediction of the lowest vertical one-electron excitation energy and the relevant molecular orbital contours and charge densities changes using density functional theory calculations. These were found to corroborate differences in acidity in the ground and excited states.