The effects of site exposure (microclimate) and forest management (thinning) on fungal-to-bacterial (F:B) respiratory ratio and N2O emission from forest floor and Ah layer samples were studied at untreated and thinned beech forests. Microclimate effects were studied by selecting sites facing north-east (NE) or south-west (SW). The F:B respiratory ratio was estimated using substrate-induced respiration in combination with inhibitors either affecting fungi or bacteria. N2O production was evaluated after moistening samples initially pre-incubated at different moisture levels to 100% of the water holding capacity (WHC). F:B respiratory ratios were significantly affected by microclimate and thinning, with site exposure having the strongest effect on fungal-to-bacterial ratio and N2O production both for the forest floor and the Ah layer. Significantly more N2O was produced from soils pre-incubated under low (15% WHC) moisture conditions as compared to soils pre-incubated under air dry (5% WHC) or wet conditions (30–60% WHC). A positive correlation between N2O emission and F:B respiratory ratio for Ah layer samples and a negative correlation between bacterial substrate induced respiration (SIR) and N2O emission for both Ah layer and forest floor samples indicated that net N2O production was the result of predominantly fungal N2O production and predominantly bacterial N2O consumption. The latter hypothesis was further supported by increased N2O emission from samples treated with bacterial inhibitor.