Mechanical pre-treatment of lignocellulosic biomass has been extensively applied in biofuel production despite its high energy requirements. To balance the consumed energy with the energy produced, careful selection and evaluation of pre-treatment parameters, equipment and desired outcome is needed. The study aims to determine optimal hay and barley straw biomass particle size in view of sugar yields, energy consumption and treatment time. The results show that there is no significant difference (p > 0.05) in sugar yields from hay biomass with particle sizes 0.25 mm, 1 mm and 10 mm. Energy requirements for the production of 1 kg of sugar from hay range from 1.8–10.7 MJ. At the same time barley straw proved to be inappropriate for sugar extraction due to low sugar yields (below 40 mg g-1 dry mass) and high energy consumption (18.5–76.2 MJ to produce 1 kg sugar). Thus, after the careful selection of biomass, mechanical pre-treatment followed by enzymatic hydrolysis can be an effective technique in biofuel production from biomass.