In Chlebowo mire (Wielkopolska region), we investigated testate amoebae in relation to ten environmental parameters in the semi-natural floating vegetation of flooded peat workings. The measured parameters included: depth to water table (DWT), ground-water pH, color, conductivity, PO4, NO3, NH4, SO4, Ca, and Mg. Detrended correspondence analysis and canonical correspondence analysis (CCA) were used to analyze relations between the composition of testate amoebae communities and those variables. In canonical correspondence analysis, DWT, pH, and Mg remained after forward-selection as the main factors characterizing the changes in testate amoebae communities along the moisture and nutrient gradients. Characteristic species of testate amoebae for the various stages of floating-mat development in the flooded peat workings were distinguished. Communities of testate amoebae along with present-day vegetation reflect the process of progressive acidification, driven mainly by Sphagnum fallax. We consider this as secondary succession, since preliminary investigations of peat stratigraphy revealed sedge peat below no more than 5–40 cm of Sphagnum peat in undisturbed parts of the mire. This study increases our understanding of relationships between testate amoebae and their habitat, which is valuable for palaeoenvironmental studies. A local transfer function was developed with the use of six models: partial least squares, maximum likelihood, modern analogue technique, weighted averaging, tolerance down-weighted averaging, and weighted averaging-partial least squares. The weighted averaging model performed the best for depth to water table (root mean square error of prediction RMSEP = 6.99) and pH (RMSEP = 0.8). Results will be used as part of a regional training set to improve palaeoenvironmental reconstructions of Sphagnum peatlands.