There is widespread interest in developing methods to investigate in situ microbial activity in subsurface environments. Novel experiments based on single borehole push–pull methods were conducted to measure in situ microbial activity at the Äspö Hard Rock Laboratory (HRL). Microbial nitrate reduction and lactate consumption were measured at in situ conditions at a depth of 450 m in the HRL tunnel. A circulation system was used to circulate ground water from the aquifer through pressure-maintaining flow cells containing coupons for biofilm growth. The system allows microbial investigations at in situ pressure, temperature and chemistry. Four experiments were conducted in which a combination of a conservative tracer, nitrate and lactate was injected into the circulation system. Rate of nitrate utilization was 5 µm h−1 without lactate and 13 µm h−1 with lactate. Lactate consumption increased from 30 to 50 µm h−1 with the addition of an exogenous electron acceptor (nitrate). Attached and unattached cells were enumerated using epifluorescence microscopy to calculate cell-specific rates of activity. The biofilm had an average cell density of 1 × 106 cells cm−2 and there was an average of 6 × 105 unattached cells mL−1 in circulation. Cell-specific rates of lactate consumption were higher than previously reported using radiotracer methods in similar environments. The differences highlight the importance of conducting microbial investigations at in situ conditions. The results demonstrate that an indigenous community of microbes survives at a depth of 450 m in the Fennoscandian shield aquifer with the potential to oxidize simple organic molecules such as lactate.