In boreal forest ecosystems, nitrogen imbalance has long been discussed as a mystery with regard to its high biomass production. Under conditions widely used for the soybean nodulation bacterium Bradyrhizobium japonicum, microbiota in forest bed soil often do not show any significant acetylene reduction. However, in a recent study, soil bacterial microbiota in an East Siberian Taiga forest bed and bacteria isolated from the soil show remarkable acetylene reduction in nitrogen-poor soft-gel medium solidified with gellan gum instead of agar. Thus, we precisely investigated optimal culture conditions including compositions of the medium; appropriate conditions for the soil microbiota were determined as follows: 0.5 g L−1d-mannitol as the carbon source, 10–15°C incubation temperature and medium pH 6.2. These culture conditions resembled the physicochemical conditions of the active layer in the East Siberian boreal forest bed on the permafrost. In fact, mannitol, which was the most appropriate carbon source, was obtained as a major sugar component of the cowberry root mats that cover the larch forest bed in this region. The most profitable concentration of d-mannitol (0.5 g L−1) for acetylene reduction was only 1/20 or 1/30 of the amount of the carbon source used in worldwide acetylene reduction assays. In a similar manner, 10–15°C, which was the most profitable incubation temperature range, was close to the reported temperature of the active layer of the bulk soil in summer. Thus, even in a 3.0 g L−1 gellan gum medium, an acetylene reduction assay under the imitated soil conditions led to clear increases in acetylene reduction of the bacterial communities tested. The N2-fixing potential of boreal forest bed soil had thus ever been evaluated unfairly low, because the world wide assay conditions were too far from the soil environments.