Beta-cyclodextrin (β-CD) is a cyclic oligosaccharide consisting of seven glucose units. β-CD is increasingly used in food research to reduce cholesterol due to its affinity for non-polar molecules such as cholesterol and as a natural additive. The purpose of this study was to evaluate the effect of curd washing in ewe’s milk cheese on the reduction in cholesterol by β-CD from pasteurized ewe’s milk Manchego cheese and the characteristics of its main components: milk, lipids, and flavor. An approximately 98.45% cholesterol reduction was observed in washed experimental cheeses that were treated by using β-CD. The remaining residual β-CD from the effect of curd washing was 0.15% in mature cheese, of the initial 1% β-CD treatment of the milk. The chemical properties (fat, moisture, and protein) did not change as a result of the curd washing with or without β-CD. The curd washing with or without β-CD on the levels of the various lipid fraction (fatty acids, triglycerides, and phospholipids) were comparable in treated and untreated cheeses. The effects of curd washing and the β-CD treatment did not significantly affect flavor components or short chain free fatty acids. The β-CD molecules were edible and nontoxic; as a result, they could be used safely in cholesterol removal processing in cheese manufacturing, improving the reduction in residual β-CD by curd washing by 85%. Therefore, the present study suggests that curd washing combined with β-CD is an effective process for cholesterol removal in Manchego cheese, preserving its desirable properties.