Se nanoparticles (NPs) with smaller size often exhibit higher antibacterial activity, thus size control of Se NPs is important to develop its application in the antibacterial field. In this study, Se NPs loaded attapulgite (APT) nanocomposites (Se/APT) were successfully prepared by a one-pot green method mediated by Aloe vera leaf extract, for which APT acts as a support to anchor Se NPs leading to the formation of small-sized and dispersed Se NPs. Structure characterization showed that the well-crystalline Se NPs with a size range of 1 ∼ 3 nm were uniformly distributed on the surface of APT nanorods. Antibacterial activities of the Se/APT nanocomposites were examined against S. aureus, and the result showed that the higher the Se loadings, the better the antibacterial activities of the nanocomposites, and the minimum inhibitory concentration of Se/APT-40% nanocomposite was up to 0.5 mg/mL. In addition, the green-synthesized nanocomposites have little cytotoxicity on mouse fibroblast cell L-929, and conversely promoted the growth and proliferation of the cells. The nanocomposites are expected to be candidates used in various antibacterial fields for preventing infections induced by S. aureus, such as suppuration of the wound.