1,2,4-Triazolo[4,3-a]pyridine (TP) is a key intermediate in pesticides, materials and medicines. The title compound 4-(((8-chloro-3-oxo-[1,2,4]triazolo[4,3-a]pyridin-2(3H)-yl)thio)methyl)benzonitrile was synthesized via three steps, and its structure was characterized by ¹H NMR, HRMS and X-ray diffraction. Single-crystal X-ray diffraction reveals that it crystallizes in the monoclinic system, space group P2₁/c. Four TP molecules in the symmetric unit are linked through the O–H···O and O–H···N hydrogen bonding interactions via two H₂O molecules along with two π-π interactions. The preliminary antifungal activity results indicated that the compound TP exhibited good activities. Theoretical calculation was carried out by DFT method using the 6-31G basis set.