To date, microwave radiation has been successfully used to support the chemical hydrolysis of organic substrates in the laboratory. There is a lack of studies on large-scale plants that would provide the basis for a reliable evaluation of this technology. The aim of the research was to determine the effectiveness of using microwave radiation to support the acidic and alkaline thermohydrolysis of lignocellulosic biomass prior to anaerobic digestion on a semi-industrial scale. Regardless of the pretreatment options, similar concentrations of dissolved organic compounds were observed, ranging from 99.0 ± 2.5 g/L to 115.0 ± 3.0 in the case of COD and from 33.9 ± 0.92 g/L to 38.2 ± 1.41 g/L for TOC. However, these values were more than twice as high as the values for the substrate without pretreatment. The degree of solubilisation was similar and ranged between 20 and 28% for both monitored indicators. The highest anaerobic digestion effects, ranging from 99 to 102 LCH4/kgFM, were achieved using a combined process consisting of 20 min of microwave heating, 0.10–0.20 g HCl/gTS dose, and alkaline thermohydrolysis. For the control sample, the value was only 78 LCH4/kgFM; for the other variants, it was between 79 and 94 LCH4/kgFM. The highest net energy gain of 3.51 kWh was achieved in the combined alkaline thermohydrolysis with NaOH doses between 0.10 and 0.20 g/gTS. The use of a prototype at the 5th technology readiness level made it possible to demonstrate that the strong technological effects of the thermohydrolysis process, as demonstrated in laboratory tests to date, do not allow for positive energy balance in most cases. This fact considerably limits the practical application of this type of solution.