The acetophenone-based 3,4-dihydropyrimidine-2(1H)-thione was synthesized by the reaction of 4-methylpent-3-en-2-one (1), 4-acetyl aniline (2) and potassium thiocyanate. The spectroscopic analysis including: FTIR, H-1-NMR, and single crystal analysis proved the structure of synthesized compound (4), with the six-membered nonplanar ring in envelope conformation. In crystal structure, the intermolecular N-H MIDLINE HORIZONTAL ELLIPSIS S and C-H MIDLINE HORIZONTAL ELLIPSIS O hydrogen bonds link the molecule in a two-dimensional manner which is parallel to (010) the plane enclosing R-2(2) (8) and R-2(2) (10) ring motifs. After that, the Hirshfeld surfaces and their related two-dimensional fingerprint plots were used for thorough investigation of intermolecular interactions. According to Hirshfeld surface analysis, the most substantial contributions to the crystal packing are from H MIDLINE HORIZONTAL ELLIPSIS H (59.5%), H MIDLINE HORIZONTAL ELLIPSIS S/S MIDLINE HORIZONTAL ELLIPSIS H (16.1%), and H MIDLINE HORIZONTAL ELLIPSIS C/C MIDLINE HORIZONTAL ELLIPSIS H (13.1%) interactions. The electronic properties and stability of the compound were investigated through density functional theory (DFT) studies using B3LYP functional and 6-31G* as a basis set. The compound 4 displayed the high chemical reactivity with chemical softness of 2.48. In comparison to the already reported known tyrosinase inhibitor, the newly synthesized derivatives exhibited almost seven-fold better inhibition of tyrosinase (IC50 = 1.97 mu M), which was further supported by molecular docking studies. The compound 4 inside the active pocket of ribonucleotide reductase (RNR) exhibited a binding energy of -19.68 kJ/mol, and with mammalian deoxy ribonucleic acid (DNA) it acts as an effective DNA groove binder with a binding energy of -21.32 kJ/mol. The results suggested further exploration of this compound at molecular level to synthesize more potential leads for the treatment of cancer.