Warmer winters in Arctic regions may melt insulating snow cover and subject soils to more freeze–thaw cycles. The effect of freeze–thaw cycles on the microbial use of low molecular weight, dissolved organic carbon (LMW-DOC) is poorly understood. In this study, soils from the Arctic heath tundra, Arctic meadow tundra and a temperate grassland were frozen to −7.5 °C and thawed once and three times. Subsequently, the mineralisation of 3 LMW-DOC substrates types (sugars, amino acids and peptides) was measured over an 8-day period and compared to controls which had not been frozen. This allowed the comparison of freeze–thaw effects between Arctic and temperate soil and between different substrates. The results showed that freeze–thaw cycles had no significant effect on C mineralisation in the Arctic tundra soils. In contrast, for the same intensity freeze–thaw cycles, a significant effect on C mineralisation was observed for all substrate types in the temperate soil although the response was substrate specific. Peptide and amino acid mineralisation were similarly affected by FT, whilst glucose had a different response. Further work is required to fully understand microbial use of LMW-DOC after freeze–thaw, yet these results suggest that relatively short freeze–thaw cycles have little effect on microbial use of LMW-DOC in Arctic tundra soils after thaw.