Unmineable coal accounts for over 90% of the world's fossil fuel resources. Fortunately, many coal seams contain indigenous microorganisms capable of utilizing the coal as a carbon source to produce secondary biogenic coalbed natural gas. However, coal bioavailability has been shown to be a significant factor that limits the extent of bioconversion. In this study, we have analyzed the rate and yield of the biogas production and assessed the gas potential, carbon balance, stable carbon isotopes, microbial communities, and microbial pathways changes resulting from the hydrogen peroxide pretreatment with the Wyoming's Powder River Basin subbituminous coal. The results showed that coal pretreated with hydrogen peroxide can significantly enhance the bioavailability of coal for enhancing the biogas production, with a peak yield of 552.6 mu mol/g coal (437.1 Scf/ton coal) at the day 184. The stable carbon isotopic analysis indicated that the delta C-13 values of the methane and carbon dioxide were much less negative than the published field data. This suggested that the enrichment or depletion of the precursor C-13 could contribute to the shift of the carbon isotopic composition in the subsequence processes. Therefore, the data should be used with cautions for interpreting genesis of the thermogenic/biogenic methane and the methanogenic pathways. The methanogenic pathways were also investigated with re-fed experiment and microbial community analysis. The results indicated that the hydrogenotrophic pathway was not active in the original inoculum became activated after the gas production. The microbial community analysis demonstrated that the obligate hydrogenotrophic methanobacterium was the most dominant methanogens in the microcosms after the gas production. This suggested that the chemical treatment of coal has impacts on the microbial structure during the subsequent methane production phase.