In this report, we present solvent assisted excited-state proton transfer coupled to the deactivation of a photo-excited 2,2′-pyridylbenzimidazole bound to a single water molecule. Experimentally, the mass-selected 1 : 1 complex was probed using two-colour resonant two-photon ionization (2C-R2PI) and UV–UV hole-burning (HB) spectroscopy in a supersonically jet-cooled molecular beam. Computationally, three structural isomers were identified as the normal, the tautomer and the proton transfer product of the PBI–H₂O complex in the excited S₁ state using B3LYP-D4/def2-TZVPP and ADC(2) (MP2)/cc-pVDZ levels of theory. The most stable form in the ground state, i.e., the normal form, was identified using the excitation spectrum in the 30 544 to 30 936 cm⁻¹ region. The 2C-R2PI spectrum showed a sudden break-off above the 0⁰₀ + 392 cm⁻¹ region, even though the Frack–Condon activity of the S₁ ← S₀ transition was measured beyond 0⁰₀ + 1000 cm⁻¹ in the HB spectrum. The intensity of the bands associated with the excited state intermolecular vibrational modes near the break-off region was found to be drastically decreased, which indicates efficient quantum mechanical tunnelling along the hydrogen transfer coordinate. The sudden disappearance of the intermolecular vibrational modes in the spectrum revealed the existence of a deactivation channel in the PBI–H₂O complex near 392–450 cm⁻¹ above the 0⁰₀ transition. The computational investigation predicted that the deactivation of the excited-state occurred via the intersection between the S₁ and S₀ states, which was associated with the proton transfer from the H₂O to the PBI molecule along the O(3)–H(4)→N(5) coordinate. The highest energy structure was identified as the point of intersection between the nπ* (S₂) and ππ* (S₁) states. The associated barrier height was experimentally determined to be 392–450 cm⁻¹, which showed a reasonable agreement with the calculated excited-state proton transfer barrier. Competing reaction channels such as dissociation and tautomerization were found to be highly energetically inaccessible.