To test the hypothesis that rotifers release one or more chemical microalgal growth inhibitors in addition to devouring the microalgal cells, the effects of different concentrations of filtered, bacteria‐free, rotifer culture filtrate (RCF) on the growth and physiological parameters of Chlorella sp., and the response of Chlorella sp. at different starting cell densities to 10% RCF, were studied. The results show that RCF significantly decreased Chlorella cell densities during the incubation, suggesting that rotifers release some chemical(s) that inhibit microalgal cell growth. Chlorella cell densities decreased with increasing RCF concentration. Increasing the initial cell density of Chlorella dispersed the inhibitory chemical(s) present in 10% RCF over more cells, reducing their effect. The results confirm that the action of the chemical(s) released by rotifers on microalgal cell growth was dependent on both the RCF concentration and the exposure time. They also demonstrate that ≥10% RCF significantly inhibited photosynthesis and respiration, which would account for some of the decreased Chlorella cell growth in the presence of RCF. Calculations based on the data indicate that the rotifer‐derived chemical(s) released hourly from each rotifer inhibits growth by 45.5 microalgal cells in addition to the rotifer predation, with a 48 h LC50 value of 18.8% RCF. Based on these results, fresh medium instead of the old culture medium was contaminated by the rotifers.