Prescribed burning is a common management strategy in peatlands that has the potential to affect soil physicochemistry, alter biogeochemical cycles and trigger changes in vegetation structure. How burning affects prokaryotic community composition across different soil profiles is not well understood. This study explored the effects of prescribed burning on the diversity of prokaryotic communities in peat soils. Soil samples were collected from Moor House Nature Reserve, UK, a long-term monitoring site initiated in 1954 subject to three burning treatments: Burning at short rotations every 10 years, burning at long rotations every 20 years and a non-burn control. Observed species richness for archaea was highest in the topsoil of the non-burn control plots and highest for bacteria in the topsoil of the non-burn control and plots under a long rotation regime. Community composition was significantly different between different burn treatments and soil depth. Archaeal community structure was shaped by NH4+ and pH in the topsoil; by Pb, moisture and Al in the 20–40 cm profile; and by total N, total C, Al, Ca, Fe and pH in the 40–60 cm profile. Bacterial community structure was shaped by NH4+, heather cover, pH and Mg in the topsoil; by Fe, K and Pb in the 20–40 cm profile; and by Al, Ca and Fe in the 40–60 cm profile. A co-occurrence network analysis revealed that the topsoil of the non-burn control plots had a more complex network structure with more positive links than those under a rotational burn, but a higher average connectivity with a higher number of negative links was observed in the long rotation 20–40 cm profile. The results provide a new insight into the response processes of soil prokaryotic communities to burning in peatland soils, providing valuable knowledge that can support the evaluation and management of ecosystem services in peatlands.