Electrokinetic potential plays an important role in textile wet processes such as dyeing, finishing, and washing. The zeta potential of cotton fiber in aqueous solution is negative and influences adsorption of surfactants having the same or opposite electrical sign. The influence of zeta potential of cotton fibers on the adsorption of two cationic surfactants, dodecyltrimethyl ammonium chloride and distearyldimethyl ammonium chloride, at pH 4, 7, and 9 was studied. Cotton threads were mercerized in NaOH solutions in a slack state. Mercerization causes changes in fine structure, morphology, and conformation of cellulose chains and lowers the negative electrokinetic potential of cotton fibers. Based on this phenomena, cotton threads were treated in NaOH solutions of various concentrations resulting in samples with different degrees of mercerization and different electrokinetic potential. The amount of adsorbed surfactant on cotton fibers was quantitatively determined from the surfactant content in solution by the two-phase titration methods. The electrokinetic potential was measured using the streaming current method by EKA. The zeta potential was calculated from the Helmholtz-Smoluchowski equation.