Ball-milled seashells, as a nano-biocomposite catalyst and natural source of CaCO3 in its aragonite microcrystalline form with fixed CO2, was optimized for the synthesis of isoamyl acetate (3-methylbutyl ethanoate) by response surface methodology with a five-level three-factor rotatable circumscribed central composite design. The seashells nano-biocomposite has proved to be an excellent heterogeneous multifunctional catalyst for the green and environmentally-benign synthesis of isoamyl acetate from acetic acid and isoamyl alcohol under solvent-free conditions. A high yield of 91% was obtained under the following optimal conditions: molar ratio of alcohol: acetic acid (1:3.7), catalyst loading (15.7 mg), the reaction temperature (98 °C), and the reaction time (219 min). The outstanding advantages of this protocol are the use of an inexpensive, naturally occurring and easily prepared nano-biocomposite material having appropriate thermal stability and without any modifications using hazardous reagents, lower catalyst loading and reaction temperature, no use of corrosive Bronsted acids as well as toxic azeotropic solvents or water adsorbents, and simplicity of the procedure.