<p>The rapid expansion of industrial activity has significantly increased sludge generation from wastewater treatment plants. Slaughterhouse sludge poses serious treatment challenges due to its high organic load, pathogenic microorganisms, and complex pollutant profile. The present work evaluates ultrasonic pretreatment as a strategy to enhance the anaerobic digestion (AD) of slaughterhouse sludge, using soluble chemical oxygen demand (sCOD) as an indicator of biogas production potential. A central composite design under response surface methodology (RSM) was employed to study the effects of ultrasonication power (223–650 W) and exposure time (13–52 min) on sCOD solubilization. The maximum sCOD of 9090 mg/L was achieved at 650 W and 32.5 min, indicating a notable enhancement in solubilization efficiency. Analysis of variance (ANOVA) confirmed the model’s robustness (R² = 0.976), with ultrasound power exerting a stronger influence than exposure time. The ultrasonic pretreatment with RSM optimized conditions increased specific biogas yield by 7.15-fold during the AD process, compared to untreated sludge. The composition of biogas (especially methane) also improved due to ultrasound pre-treatment, possibly due to accelerated hydrolysis, particle size reduction, and enhanced biodegradability. The RSM findings were validated with the experimental biogas yields obtained from ultrasound-pre-treated Slaughterhouse sludge samples. Thus, the present work demonstrates ultrasonication as a promising and environmentally friendly pretreatment method, with strong potential to optimize industrial-scale biogas recovery from high-strength organic wastes such as slaughterhouse sludge.</p>