The presence of microbial contamination in the wort during the fermentation process results in damages of billions of dollars per year all around the world and promotes the industry dependence of chemicals and antibiotics to control the contamination. For these reasons, this study aimed to use the electron beam to sterilise wort from sugarcane molasses and investigate its bioethanol fermentation. Five treatments (T0 – T4) were carried out using ionizing doses of radiation through the electron accelerator: (0, 10, 20, 40, and 80 kGy) and a positive control (T5-steam by autoclave) were performed. It was evaluated total mesophiles, total bacteria, sugars, phenolics, flavonoids, 5-hidroximetylfurfural, and Furfural. After the irradiation process, it was conducted an alcoholic fermentation assay using baker’s yeast Saccharomyces cerevisiae. It was not observed inversion of sugars and formation of the inhibitory by-products flavonoids, furfural, and 5-hidroximetylfurfural, with exception of the phenolic compounds. Only T4 and T5 were able to sterilise the wort. However, T3 was able to eliminate > 99.99% of the microorganisms. In the fermentation T2 promoted the best ethanol yield and productivity among the irradiated treatments, evidencing the possibility of electron beam use in the wort treatment prior to fermentation which may allow a reduction in losses caused by microbial contamination, besides the possibility of promoting fermentation yield and productivity increase. The operation cost for applying the e-beam with a dose of 20 kGy in a standard ethanol plant was estimated at US$ 0.014 per m3 of wort.