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Boreal peatlands are key high-latitude ecosystem types and act as a carbon (C) sink storing an estimated 25% of the world's soil C. These environments are currently seeing the most substantial changing climate, especially during the winter. CO₂ emissions during the winter can correspond to 80% of the growing season's net CO₂ assimilation. Yet, our conceptual understanding of the controls on microbial metabolic activity in peat soils at temperatures ≤0 °C is poor. We used stable isotope probing of peat samples and tracked the fate of ¹³C-glucose using ¹³C-NMR. We show that microorganisms in frozen boreal peat soils utilize monomeric C-substrates to sustain both catabolic and anabolic metabolism at temperatures down to −5 °C. The ¹³C-substrate was transformed into ¹³C–CO₂, different metabolites, and incorporated into membrane phospholipid fatty acids. The 16S rRNA-based community analyses revealed the activity at −3 °C changes the composition of the bacterial community over relevant timescales. Below 0 °C, small temperature changes have strong effects on process rates and small differences in winter soil temperature may affect C dynamics of northern peatlands. Understanding biological processes at low and below zero temperatures are central for the overall functioning of these systems representing one of the world's major soil C pools.