The lateral membrane of the cochlear outer hair cell (OHC) is the site of a membrane-based motor that powers OHC electromotility, enabling amplification and fine-tuning of auditory signals. The OHC membrane protein prestin plays a central role in this process. We have previously shown that membrane cholesterol modulates the peak voltage of prestin-associated nonlinear capacitance in vivo and in vitro. The present study explores the effects of membrane cholesterol and docosahexaenoic acid content on the peak and magnitude of prestin-associated charge movement in a human embryonic kidney (HEK 293) cell model. Increasing membrane cholesterol results in a hyperpolarizing shift in the peak voltage of the nonlinear capacitance (Vpkc) and a decrease in the total charge movement. Both measures depend linearly on membrane cholesterol concentration. Incubation of cholesterol-loaded cells in cholesterol-free media partially restores the Vpkc toward normal values but does not have a compensatory effect on the total charge movement. Decreasing membrane cholesterol results in a depolarizing shift in Vpkc that is restored toward normal values upon incubation in cholesterol-free media. However, cholesterol depletion does not alter the magnitude of charge movement. In contrast, increasing membrane docosahexaenoic acid results in a hyperpolarizing shift in Vpkc that is accompanied by an increase in total charge movement. Our results quantify the relation between membrane cholesterol concentration and prestin-associated charge movement and enhance our understanding of how membrane composition modulates prestin function.