Plant pigments gained popularity over synthetic dyes because of safety issues regarding the impact on health and environment. The present paper deals with optimization of anthocyanins extraction from Paeonia officinalis L. flowers and their application in cotton eco-dyeing processes. The results on extraction and mathematical models indicate improved extraction by ultrasonication at high solvent/solid ratio (50/1) compared to that by maceration. Among the investigated extraction solvents, the most efficient was 70% ethanol. Cotton dyeing was performed via conventional procedure and ultrasonication, in the presence and absence of classic mordant (copper sulphate) and biomordants (tannic and citric acids). A successful dyeing was obtained according to ATR-FTIR analysis of mordanted dyed samples. The colour properties of dyed cellulosic substrates as determined by CIELAB system showed higher redness values and large positive differences in chroma in samples dyed with peony extract by exhaustion in presence of high concentration of citric acid.