Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) is a promising thermoelectric material with (bi)polaron-type transport properties. Dedoping PEDOT:PSS by simply controlling film thickness and the annealing temperature under high relative humidity led to partial reduction and a conformational change in the polymer that improved both the electrical conductivity and the Seebeck coefficient. When the film was hydrothermally annealed at 170 °C, its Seebeck coefficient was enhanced from 13.8 to 42.3 μV K-1. Water was the reducing agent and at an optimum annealing temperature of 130 °C facilitated separation of reduced PEDOT and protonated PSS chain, resulting in an improved power factor (PF) of 16.9 μW m-1 K-2 and a color change in the annealed PEDOT:PSS films from light to dark blue. The acid-treated PEDOT:PSS film followed by hydrothermal annealing exhibited a conductivity of 1241 ± 272 S cm-1 and 28.7 ± 2.1 μV K-1, and the corresponding PF was 103.9 μW m-1 K-2. These results show the possibility of tuning the thermoelectric properties of PEDOT:PSS films for biomedical applications without the need of toxic or expensive additives.