Many synthetic DNA minor groove binders exhibit a strong increase in fluorescence when bound to DNA. The pharmaceutical-relevant berenil (diminazene aceturate) is an exception with an extremely low fluorescence quantum yield (on the order of 10–4). We investigate the ultrafast excited-state dynamics of this triazene by femtosecond time-resolved fluorescence experiments in water, ethylene glycol, and buffer and bound to the enzyme β-trypsin, the minor groove of AT-rich DNA, and G-quadruplex DNA. Ab initio calculations provide additional mechanistic insight. The complementing studies unveil that the excited-state motion initiated by ππ* excitation occurs in two phases: a subpicosecond phase associated with the lengthening of the central N═N double bond, followed by a bicycle-pedal-type motion of the triazene bridge, which is almost volume-conserving and can proceed efficiently within only a few picoseconds even under spatially confined conditions. Our results elucidate the excited-state relaxation mechanism of aromatic triazenes and explain the modest sensitivity of the fluorescence quantum yield of berenil even when it is bound to various biomolecules.