This study investigated the effect of granular activated carbon (GAC) on the digestion of blackwater collected from different collection systems, by monitoring the biochemical methane production (BMP), adsorption of molecules to GAC and their impacts on the microbial community. Without GAC-amendment, BMP reached 35.6, 42.6 and 50.4% in 1L, 5L and 9L water-flushed blackwater, respectively. When 33.3 gL-1 GAC was added to the cultures, methane potential increased up to 53.1% in 1L water-flushed blackwater, while in 5L and 9L water-flushed conditions the BMP drastically decreased to 16.1 and 9.6%, respectively. The concentration of volatile fatty acids (VFA) in 5L and 9L water-flushed blackwater with GAC-amended cultures was not enhanced, in contrast with 1L water-flushed blackwater. Further tests showed 29.8% (±1.9%) of VFA and 86.0% of soluble chemical oxygen demand were removed by GAC adsorption in 9L water-flushed blackwater. A decrease in biomass density in 5L and 9L GAC-amended cultures was also observed, corroborated by a significant decrease in gene copy numbers of methanogenic archaeal communities. This study gives an insight on the effect of GAC on different strengths of blackwater, which is of relevance for further tests of long-time and full-scale application.