The combined effects of three key ozonation process parameters on the integrated ozonation-enzymatic hydrolysis pretreatment of sugarcane bagasse (SCB) were investigated, with emphasis on the relationship between sugar release and ozone consumption. A lab-scale fixed bed reactor was employed for ozonation at varying ozone doses (50, 75 and 100 mg O3/g SCB), particle sizes (420, 710 and 1000 µm) and moisture contents (30, 45 and 60% w/w) in multifactorial experiments, keeping a residence time of 30 min. The ozonated SCB showed a reduction in the content of acid-insoluble lignin from 26.6 down to 19.1% w/w, while those of cellulose and hemicellulose were retained above 45.5 and 13.6% w/w, with recoveries of 100−89.9 and 83.5−72.7%, respectively. Ozone-assisted enzymatic hydrolysis allowed attaining glucose and xylose yields as high as 45.0 and 37.8%, respectively. The sugars released/ozone expended ratio ranged between 2.3 and 5.7 g sugars/g O3, being the higher value achieved with an applied ozone input of 50 mg O3/g SCB and SCB with 420 µm particle size and 60% moisture. Such operating conditions led to efficient ozone utilization (<2% unreacted ozone) with a yield of 0.29 g sugars/g SCB. Overall, the amount of sugars released relative to the ozone consumed was improved, entailing an estimated cost of ozonation of USD 34.7/ton of SCB, which could enhance the profitability of the process.