Here, the role of the voltage waveform and the electrolytic ions in the actuation mechanism of Poly (3,4-ethylene dioxythiophene) polystyrene sulfonate (PEDOT:PSS)/bacterial cellulose bilayer actuator is reported. PEDOT:PSS/bacterial cellulose-based bilayer actuator was fabricated, and its mechanism under a square and triangular wave potential at multiple frequencies was studied. The actuation was performed with HCL, KCl, and LiCl electrolytes. The deflection of the actuator was found to be dependent on the time period of the voltage waveform. The deflection for opposite biases showed frequency-dependent asymmetry. Further, the actuator deflection was found to be related to the hydrodynamic size of cations present in electrolytes. The results indicate a cation-dominated actuation mechanism for PEDOT:PSS/bacterial cellulose bi-layer actuator. © 2023 John Wiley & Sons Ltd.