Anaerobic digestion (AD) is a well-established technology for producing biogas in wastewater treatment plants using organic-rich sludges. As part of the AD process, the hydrolysis step is the rate-limiting factor because of its critical role in breaking down complex organic materials into simpler, more readily digestible compounds. In this study, thickened waste-activated sludge (TWAS) samples taken from the Saskatoon Wastewater Treatment Plant (SWTP) were used to investigate the effects of ultrasonic (US) pretreatment on the AD process for biogas production. The US pretreatment facilitates hydrolysis and increases the solubilization of chemical oxygen demand (COD) during AD. The Response Surface Methodology (RSM) based on a Central Composite Design (CCD) method was employed to optimize US pretreatment operational parameters by considering disintegration time and amplitude as input variables and cumulative biogas production as the response. After 30 days of the AD process, the optimum US treatment resulted in the highest biogas production of 244.9 mL/g VS (volatile solids) using 47% amplitude and 15.5 min disintegration time. In contrast, the lowest production rate of 160.5 mL/g VS was found for the control sample without any pretreatment under the same conditions. By optimizing AD performance through US pretreatment, these findings provide valuable insight into managing municipal wastewater treatment plant AD processes. This study may inform potential improvements in AD performance in Canada and global implications for providing a more sustainable waste management and energy recovery process.