This work investigated how substrate-specific pretreatment, operating temperature, and organic loading rate (OLR) influenced the performance of semi-continuous anaerobic co-digestion using the organic fraction of municipal solid waste (OFMSW), thickened waste activated sludge (TWAS), and rice straw (RS). Four laboratory-scale reactors were operated under mesophilic (35 °C) and thermophilic (55 °C) conditions using untreated or selectively pretreated co-substrates. TWAS was subjected to thermo-alkaline pretreatment, whereas RS was pretreated with hydrogen peroxide (H₂O₂). The reactors were operated at OLRs of 2, 3, and 4 g VS L⁻¹ d⁻¹, corresponding to hydraulic retention times of 16, 11, and 8 d, respectively. Thermophilic digestion combined with substrate-specific pretreatment showed the highest overall biogas production performance. At the highest organic loading rate (4 g VS L⁻¹ d⁻¹), Reactor C produced the maximum volumetric biogas yield (2.13 ± 0.12 m³ m⁻³ d⁻¹) while maintaining a COD removal efficiency of 73%. It also produced the highest final cumulative biogas volume of 31,886 mL, representing increases of 18.6%, 25.0%, and 23.2% compared with Reactors A, B, and D, respectively. Stepwise increases in OLR caused short-term fluctuations in pH and volatile fatty acid concentrations. However, these transient responses were consistent with microbial acclimation and did not indicate sustained process inhibition. A preliminary energy assessment further indicated that the incremental recoverable energy associated with pretreatment-enhanced biogas production was substantially greater under thermophilic than mesophilic conditions. Overall, integrating substrate-specific pretreatment with thermophilic semi-continuous co-digestion improved organic matter conversion while sustaining biogas production under progressively increasing organic loading conditions.