The effects of steam and sulfuric acid pretreatment on anaerobic digestion (AD) of whiskey byproducts (including draff, thin and thick stillage) were investigated in order to improve the digestion performance under both mesophilic and thermophilic temperatures. The results of biomethane potential assays suggested that thermophilic AD facilitated the release of free ammonia (ca. 1200 mg/L) from byproducts, resulting in strong ammonia inhibition and volatile fatty acid accumulation. In contrast, no free ammonia inhibition (ca. 700 mg/L) was observed under mesophilic AD; the methane yield from mesophilic AD was between 375.3 ± 13.6 mL/g volatile solid (VS; acid-treated sample) and 389.1 ± 8.5 mL/g VS (untreated sample). Although acid pretreatment (2% acid under 135 °C for 15 min) did not improve the methane yield from mesophilic AD, it reduced the digestion time by 14.3% compared to that of the untreated sample. Microbial community analysis showed that irrelevant of pretreatment, hydrogenotrophic methanogens of Methanobrevibacter (28.9%–49.8% in abundance) and Methanoculleus (26.0%–55.9% in abundance) were the dominant archaeal genus under mesophilic AD. In comparison, hydrogenotrophic Methanothermobacter (over 97% in abundance) were dominant in thermophilic AD. This study could be exploited to aid in decarbonizing the whiskey industry by optimizing the biogas process in a circular economy system.