This study analyzed the effects of thermohydrolysis on the anaerobic conversion efficiency of lignocellulosic biomass, comparing conventional and microwave heating methods. The research aimed to identify the optimal temperature and duration for biomass pre-treatment to maximize biogas output. Four temperatures (100 °C, 130 °C, 150 °C, and 180 °C) and six durations (10, 15, 20, 25, 30, and 40 min) were tested. The results showed that microwave heating increased biogas production compared to conventional heating at the same temperatures and durations. At 150 °C, microwave heating for 20 min produced 1184 ± 18 NmL/gVS of biogas, which was 16% more than the 1024 ± 25 NmL/gVS achieved through conventional heating. Statistically significant differences in biogas output between microwave and conventional heating were observed at 130 °C, 150 °C, and 180 °C, with the greatest difference recorded between 130 °C and 150 °C: 13% for conventional heating and 18% for microwave heating. Notably, increasing the temperature from 150 °C to 180 °C did not result in a statistically significant rise in biogas production. The energy balance analysis revealed that microwave heating, despite its lower efficiency compared to conventional heating, resulted in higher net energy gains. The most favorable energy balance for microwave heating was observed at 150 °C, with a net gain of 170.8 Wh/kg, while conventional heating at the same temperature achieved a gain of 126.2 Wh/kg. Microwave heating became cost-effective starting from 130 °C, yielding an energy surplus of 18.2 Wh/kg. The maximum energy output from microwave conditioning was 426 Wh/kg at 150 °C, which was 158 Wh/kg higher than conventional heating. These findings suggest that microwave thermohydrolysis, particularly at 150 °C for 20 min, enhances both biogas production and energy efficiency compared to conventional methods. The results highlight the potential of microwave pre-treatment as an effective strategy to boost methane fermentation yields, especially at temperatures above 130 °C.