Turkey litter waste is lignocellulosic waste that can be sustainably used as an energy source through anaerobic digestion (AD). The 16S ribosomal RNA technique helps to unravel microbial diversity and predominant metabolic pathways. The assays were performed in 600-mL-glass bottles with 400 mL volume, for 60 days at 37 °C. The study evaluated the physicochemical parameters, the composition of the microbiota, and the functional inference in AD of different concentrations of turkey litter (T) using two inocula: granular inoculum (S) and commercial inoculum (B). The highest accumulated methane production (633 mL CH4·L-1) was observed in the test containing 25.5 g VS·L-1 of turkey litter with the addition of the two inocula (T3BS). In tests without inoculum (T3) and with commercial inoculum (T3B), there was an accumulation of acids and consequent inhibition of methane production 239 mL CH4·L-1 and 389 mL CH4·L-1, respectively. Bacteroidota, Firmicutes, and Actinobacteria were the main phyla identified. The presence of archaea Methanobacterium, Methanocorpusculum, and Methanolinea highlighted the hydrogenotrophic metabolic pathway in T3BS. Functional prediction showed enzymes involved in three metabolic pathways in turkey litter biodigestion: acetotrophic, hydrogenotrophic, and methylotrophic methanogenesis. The predominant hydrogenotrophic pathway can be observed by analyzing the microbiota, archaea involved in this specific pathway, genes involved, and relative acid consumption for T3S and T3BS samples with higher methane production. Molecular tools help to understand the main groups of microorganisms and metabolic pathways involved in turkey litter AD, such as the use of different inocula, allowing the development of strategies for the sustainable disposal of turkey litter.