To mitigate the diversion of food matrices to the fuel sector, repurposing agro-industrial waste through second-generation ethanol (2G) production from lignocellulosic sources emerges as an efficient strategy. This approach can be accomplished by direct conversion, which entails involves genetically modifying Saccharomyces cerevisiae for heterologous expression of cellulases, facilitating the breakdown of complex lignocellulosic structures into fermentable sugars for ethanol synthesis. This study evaluates the performance of three genetically modified S. cerevisiae strains (A, B, and C) expressing endoglucanase and β-glucosidase, in comparison to their parental counterpart. Cultivation occurred in YPD medium (1% yeast extract, 2% peptone, and 6% glucose; m/v), supplemented with 2% carboxymethylcellulose (m/v) at 35 °C in anaerobiosis for up to 24 h, with periodic sample collection. Analytical procedures involved HPLC-RI for quantifying glucose and ethanol concentrations. Notably, the modified strains exhibited superior ethanol production within the initial six hours, confirming the success genetic modification. However, beyond this period, the parental strain matched the ethanol production of strain B and surpassed strains A and C. At 24 hours, the parental strain achieved an ethanol yield of 0.40 g/g, while modified strains achieved 0.41, 0.35, and 0.36 g/g, respectively. Despite the initial success, operational adjustments, such as enhanced aeration and agitation at the process outset, are necessary to stimulate greater enzyme production. In summary, the findings suggest an interplay of genetic and environmental factors influencing ethanol production. Further research into regulatory mechanisms and optimization of operational conditions is crucial to fully harness the potential of genetically modified yeast strains in lignocellulosic ethanol production.