1. Cutover peatlands are valuable as habitat for rare species and as carbon sinks. However, assessing peat accumulation is complicated. Approaches such as using biological and physico-chemical indicators may represent an alternative for managers. 2. In order to assess the potential of biological and physico-chemical parameters as restoration indicators, we studied the organic matter (OM), testate amoebae and bacteria in peat profiles from a cutover bog. We selected four regeneration stages and an unexploited area of the same peatland. Living Sphagnum was analysed for testate amoebae. Physico-chemical parameters were analysed on near-surface peat profiles. 3. Contrasting micromorphological and biochemical signatures of peat OM were observed along the profiles. Regenerating and natural peat profiles differ with respect to C/N ratio and OM degradation. 4. The OM composition of the newly regenerated peat also differed along the regeneration sequence. Peats from the recent regenerated sites were dominated by Sphagnum-derived tissues and were characterised by lesser carbohydrate preservation and a high bacterial biomass in comparison to the peat from older regenerating sites which showed a heterogeneous botanical composition and lower bacterial biomass. 5. Moss OM inputs are characterised by hemicellulosic mannose and galactose, while sedge inputs are characterised by hemicellulosic xylose and arabinose. Additional indicators of OM alteration included the differential biodegradation of cellulose and hemicellulose. 6. Testate amoeba communities changed from the early to the advanced stages of regeneration suggesting a shift from wet and mesotrophic conditions to drier and more acidic conditions. Species richness and diversity increased but density declined from the early to the late regeneration stage and the unexploited site. Biomass and the average size of species declined over the regeneration sequence but were higher in the unexploited site. 7. The spontaneous secondary succession in the studied cutover bog leads to an ecosystem similar to that of the intact reference site in terms of community structure while OM characteristics and testate amoebae continue to reflect disturbances associated with peat harvesting. This combination of biological and physico-chemical indicators provides a complete assessment of the present and recent past ecological conditions. Such an approach has practical applications for the management of cutover peatlands.