Fractured rocks of impact craters may be suitable hosts for deep microbial communities on Earth and potentially other terrestrial planets, yet direct evidence remains elusive. Here, we present a study of the largest crater of Europe, the Devonian Siljan structure, showing that impact structures can be important unexplored hosts for long-term deep microbial activity. Secondary carbonate minerals dated to 80 ± 5 to 22 ± 3 million years, and thus postdating the impact by more than 300 million years, have isotopic signatures revealing both microbial methanogenesis and anaerobic oxidation of methane in the bedrock. Hydrocarbons mobilized from matured shale source rocks were utilized by subsurface microorganisms, leading to accumulation of microbial methane mixed with a thermogenic and possibly a minor abiotic gas fraction beneath a sedimentary cap rock at the crater rim. These new insights into crater hosted gas accumulation and microbial activity have implications for understanding the astrobiological consequences of impacts.