Dead wood is initially a nitrogen (N) poor substrate, where the N content increases with decay, partly due to biological N2 fixation, but the drivers of the N accumulation are poorly known. We quantified the rate of N2 fixation in decaying Norway spruce logs of different decay stages and studied the potential regulators of the N2-fixation activity. The average rate for acetylene reduction in the decaying wood was 7.5 nmol ethylene g−1d−1, which corresponds to 52.9 μg N kg−1d−1. The number of nifH copies (g−1 dry matter) was higher at the later decay stages, but no correlation between the copy number and the in vitro N2 fixation rate was found. All recovered nifH sequences were assigned to the order Rhizobiales, and therein mostly (60%) to methane oxidizing genera. We confirm that nitrogen fixing methanotrophs are present in all the wood decay phases and suggest that their interaction between methane producing organisms in decaying wood should be further studied. •N2 fixing bacteria are active in decaying wood.•The number of nifH copies was higher at later decay stages.•No correlation between nifH copy number and in vitro N2 fixation rate was found.•60% of the nifH sequences were assigned to methane oxidizing bacteria.