Abstract <p><b>Objective:</b> This study aims to design a small library of novel 2,5-disubstituted 1,3,4-thiadiazole derivatives and evaluate their antioxidant and acetylcholinesterase (AChE) inhibitory activities. <b>Methods:</b> A series of 2,5-disubstituted 1,3,4-thiadiazole derivatives were designed, synthesized <i>via</i> a convenient synthetic route, and assessed for their AChE inhibitory activity against electric eel AChE using Ellman’s method. Their antioxidant potential was evaluated <i>via</i> the DPPH free radical scavenging assay. The structures of the synthesized compounds were characterized using FT-IR, ¹H, ¹³C NMR spectroscopy, mass spectrometry, and elemental analysis. Molecular docking and molecular dynamics (MD) simulations were performed to identify potential AChE inhibitors. <b>Results and Discussion:</b> The results demonstrated that compounds (<b>IVe</b>), (<b>IVj</b>), and (<b>IVg</b>) exhibited significant antioxidant activity at 100 µg/mL compared to the reference standard. The AChE inhibition assay revealed that compounds (<b>IVe</b>), (<b>IVj</b>), (<b>IVg</b>), and (<b>IVc</b>) displayed potent inhibitory effects at a concentration of 0.004 M. Kinetic studies elucidated the mode of enzyme inhibition, showing that compound (<b>IVe</b>) exhibited a mixed-type inhibition mechanism, similar to Donepezil. Docking studies predicted moderate to strong binding affinities for all synthesized compounds (<b>IVa–IVj</b>) compared to the reference drug. Among them, compound (<b>IVe</b>) demonstrated the lowest docking energy (–10.549 kcal/mol), suggesting the highest anticholinesterase activity. MD simulations further confirmed the stable binding interactions of compound (<b>IVe</b>) within the active site of AChE. <b>Conclusions:</b> These findings suggest that compound (<b>IVe</b>) is a promising lead structure for the development of novel AChE inhibitors for the treatment of Alzheimer’s disease (AD).</p>