<i>Objectives:</i> Preservatives are indispensable components of aqueous multidose topical formulations. The purpose of our study was to investigate the effects of two representative preservatives, benzalkonium chloride (BKC) and potassium sorbate (PS), on rabbit tracheal ciliary beat frequency (CBF). <i>Methods:</i> Rabbit tracheal ciliated cell culture was established and CBF was determined using high-speed digital imaging methods. The effects of preservatives at different concentrations on CBF were observed over a 10-min exposure period. <i>Results:</i> BKC induced inhibition of CBF in a concentration-dependent manner. Ciliary beating was stopped by 0.01% BKC after 5 min of exposure. A low concentration of PS (0.12%) only resulted in a mild decrease in CBF during a 10-min exposure. The CBF decreased by 13.0% from baseline after 10 min. However, there was no statistically significant difference compared with the corresponding control condition. Application of 0.24, 0.48 and 0.96% of PS to rabbit tracheal cells resulted in an increase in CBF, with an increase of 105 ± 9.8, 107.6 ± 4.0, and 117.1 ± 9.5% relative to baseline CBF after 10 min of exposure, respectively. <i>Conclusions:</i> PS could be considered as a safer and more promising preservative than BKC for use in topical formulations.