•Synthesis of alkyl substituted thiourea and its characterization.•Single crystal XRD analysis.•In-Vitro carbonic anhydrase inhibition studies.•Computational studies including DFT, Molecular docking and ADMET Analysis. Carbonic anhydrases (CAs) representing the class of metalloenzymes are expressed widely and play a crucial role in various physiological and pathological processes. The Alkyl substituted Acyl Thiourea was synthesized and characterized by NMR spectroscopic technique. Single crystal X-ray diffraction was applied to determine the molecular and crystal structures. Synthesized compound crystallized in monoclinic system P 21/c space group. Intermolecular N–H···S hydrogen bonds and Intramolecular N–H···O hydrogen bond were found in crystal structure. H … H interactions have major contributions in crystal packing as depicted by Hirshfeld surface analysis. No large cavity was observed during void volume analysis in the crystal packing. Stabilization was dominated via the dispersion energy contributions in the synthesized compound. In Vitro analysis showed that synthesized compound inhibited the carbonic anhydrase activity to an appreciable extent. Quantum mechanical descriptors were calculated using DFT studies. In molecular docking, reactive parts of the synthesized molecule contributed in forming the hydrogen bond with receptor protein target. Finally, ADMET Analysis were performed to assess the drug-like behavior of synthesized compound. Carbonic Anhydrase enzyme inhibitor of pharmaceutical significance can be developed using the synthesized molecule ES-2 as a lead molecule.