ABSTRACT Microbial contamination of the wort during the fermentation process causes significant losses in ethanol production worldwide and creates a dependence of the industry on chemicals and antibiotics to control contamination. Therefore, this study used electron beam (e-beam) to disinfect wort from sugarcane (Saccharum officinarum L.) molasses and investigate the bioethanol fermentation. Four treatments (T0 – T3) were carried out using ionizing doses of radiation through the electron accelerator: 0 (control), 10, 20, and 40 kGy. Total mesophiles, total bacteria, sucrose, glucose, fructose, phenolics, flavonoids, hydroxymethylfurfural (5-HMF), and Furfural were measured. An alcoholic fermentation assay was performed after the irradiation process. The irradiated treatments showed no inversion of sugars and formation of the inhibitory by-products flavonoids, furfural and 5-HMF, except for the phenolic compounds. The lower dose tested (10 kGy) reduced more than 99.9 % of the total mesophiles and more than 99.99 % of the total bacteria in the substrate. In the fermentation, the irradiated worts presented similar (p > 0.05) yields (92, 93, and 94 %) and ethanol productivity levels (0.89, 0.88, and 0.87 g L–1 h–1, for T1, T2, and T3 respectively). However, all treatments presented higher yields and productivity (p < 0.05) when compared to the control (88 % and 0.85 g L–1 h–1), highlighting the possible use of e-beam in wort fermentation at a lower dose (10 kGy). This allows reduction in losses caused by microbial contamination, besides increasing fermentation yield and productivity with lower energy consumption.