<p><i>Euglena gracilis</i> is a metabolically versatile microalga capable of utilizing C₂ carbon sources such as ethanol to enhance biomass and paramylon production. However, large-scale non-axenic cultivation remains challenging due to microbial contamination, which can reduce yields and increase operational costs. Here, we evaluated ethanol as a dual-purpose input under non-axenic mixotrophic conditions, providing an assimilable carbon source while exerting contamination pressure that supports stable cultivation. Response Surface Methodology (RSM) was applied to optimize ethanol concentration and initial medium pH, identifying 0.58% (v/v) ethanol and an initial pH of 4.46 as optimal conditions. Under these conditions, biomass and paramylon yields reached 2.48 g L^-1 and 2.00 g L^-1, respectively, representing approximately 12-fold and 67-fold increases compared with the ethanol-free control (0.20 g L^-1 biomass; 0.03 g L^-1 paramylon). Paramylon content reached 84% of dry biomass, exceeding values commonly reported for axenic or heterotrophic systems using other carbon sources. Importantly, GC-based ethanol measurements with matched cell-free controls confirmed that ethanol depletion under the optimized condition was dominated by net biotic consumption rather than abiotic volatilization, supporting ethanol’s role as an effective co-substrate in this system. Overall, these results demonstrate that low-dose ethanol, optimized via RSM, can simultaneously maximize product yield and facilitate robust non-axenic cultivation, strengthening the feasibility of scalable <i>E. gracilis</i> biorefinery processes aligned with circular bioeconomy objectives.</p> Graphical Abstract <p></p>