The impact of a two-stage anaerobic digestion (AD) system integrating a dark fermentation (DF) step prior to AD, referred to as DF-AD2, on the organic matter (OM) quality and the microbial communities in anaerobic digestates was investigated. Two treatment routes (one-stage AD (AD1) and DF-AD2) were compared by advanced characterization using the same feedstock and treatment duration. The DF-AD2 improved the percentage of CH4 during AD2 by 8.3 % and the volatile solids removal by 6.8 % compared to AD1. The DF step increased the dissolved OM and mineralized nitrogen after AD despite similar OM complexity and predicted carbon mineralization in soils. Moreover, respirometry tests related the enhanced bioaccessibility of DF effluent to greater biological activity (126.3 ± 5.8 mg O2) compared to the substrate (51.1 ± 5.8 mg O2). Nonetheless, DF-AD2 did not impact the biological stability of digestates (32.09 ± 1.1 and 30.2 ± 1.5 mg O2 for AD1 and DF-AD2, respectively). Low-stress operational conditions of the tests might smooth the DF-AD2 effect on digestate biological stability and microbial communities. Archaea varied after DF but homogenized during AD2, with the genus Methanosarcina comprising 71–79 % of the relative abundance. Concurrently, the orders Bacteroidales, Spirochaetales and Cloacimonadales dominated Bacteria in both AD1 and AD2. Overall, this study evidence that a DF-AD2 system is a feasible way to improve both OM removal and the nitrogen fertilizing value of digestates, without hindering digestate biological stability or microbial communities. However, optimizing operational parameters and pre-treatment processes may be necessary to enhance the system's energy output.