Acid compost tea (CT) is an acidic liquid organic fertilizer, which can be used for topdressing throughout the period of cotton growth in drip irrigation cotton fields with calcareous soil. However, its effects on soil bacteria and cotton roots are not clear. This study investigated the response of cotton roots and rhizosphere bacteria to CT application. Compared with organic fertilizer, the total root length, surface area, and volume were found to increase by 18.9%, 19.9%, and 20.7% upon CT treatment, respectively. However, the abundance of rhizosphere bacteria decreased upon CT treatment. The relative abundance of bacteria associated with root growth and rhizosphere environmental health, such as Micrococcaceae, Nocardioidaceae, and Sphingomonadaceae, increased significantly, whereas the relative abundance of oligotrophic bacteria, such as Methylmirabilota, decreased. Random forest distribution showed that the cotton root was the main factor affecting rhizosphere bacterial abundance and diversity, while soil properties (pH, total nitrogen, and organic carbon) and roots affect the relative abundance of some species together. The gene expression associated with metabolism, environmental adaptation, and immune system in rhizosphere bacteria increased. In conclusion, CT can improve soil properties and promote the growth of cotton roots, and cotton roots in turn exert regulatory effects to promote the growth of beneficial bacteria in the rhizosphere and affect their functions. CT has considerable application potential for the sustainable development of cotton fields with calcareous soil.