<p>This study investigates the volumetric and acoustic properties of acetylcholine chloride in aqueous solutions by measuring density and speed of sound at molalities up to 0.6&#xa0;mol·kg⁻¹ and temperatures from 293.15&#xa0;K to 313.15&#xa0;K. From these measurements, thermodynamic parameters, including apparent molar volumes and apparent molar isentropic compressibilities, are derived, along with their limiting values at infinite dilution. The limiting apparent molar volume shows only a slight temperature dependence, whereas the limiting apparent molar isentropic compressibility exhibits a more pronounced temperature dependence. In addition, the experimental data are used to evaluate Hepler’s coefficient, and the hydration number is calculated using the Passynski method, indicating high hydration at lower temperatures and systematic changes with increasing temperature. To place the results in a broader context, comparative analyses with choline chloride, the biological precursor of acetylcholine, are conducted to demonstrate how acetylation modifies biochemical properties and solute–water interactions. To the best of our knowledge, this work provides the first systematic density and speed of sound dataset for aqueous acetylcholine chloride in water, delivering reference volumetric and acoustic properties for quantifying hydration and solute–water interactions of a physiologically important cholinergic molecule.</p>