<p>A pretreatment step is essential to improve the enzymatic digestibility of biomass, and in recent years, hydrodynamic cavitation (HC) has emerged as a promising alternative. However, while HC has been primarily studied for pretreatment, its application to directly assist the enzymatic hydrolysis process remains unexplored. In this work, a novel approach was introduced, and the effect of HC on the enzymatic hydrolysis of sugarcane bagasse was evaluated by performing a face centered central composite design with enzyme loading and temperature as variables. The analysis of the design results and the process optimization were aided by Design-Expert and Statistica software. Additionally, a sequential strategy involving HC-assisted enzymatic hydrolysis was implemented through four cycles of fed-batch processing. Under optimized conditions (enzyme loading of 18 FPU/g and temperature of 50&#xa0;°C), glucan and xylan hydrolysis yields of 91% and 77%, respectively, were achieved after 12&#xa0;h. These yields surpassed those obtained in control experiments performed with ultrasound-assisted hydrolysis. In fed-batch process, the obtained sugar concentrations were increased 5–6&#xa0;times in the hydrolysate compared to the values obtained in simple batch mode. The results demonstrate HC as an innovative and effective alternative for enhancing enzymatic hydrolysis.</p> Graphical abstract <p></p>