The influence of non-ionogenic surfactants, i.e., Tween-20, Triton X-100 and PEG-10 000, on the response of cholinesterase-based potentiometric biosensors and their sensitivity towards reversible and irreversible inhibitors were investigated. Acetyl-and butyrylcholinesterases were immobilized on nylon, cellulose nitrate films and tracing paper and were introduced into an assembly of potentiometric biosensors. The effect of surface-active compounds depends on the hydrophilic properties and porosity of the enzyme support material and the inhibition mechanism. In the range 0.002–0.3% m/v the surfactants show a reversible inhibiting effect on biosensor response. At lower concentrations (down to 10–4% m/v) the surfactants alter the analytical characteristics of reversible and irreversible inhibitor determination. The use of surface-active additives improves the biosensor selectivity in multi-component media.