Macrophages are the host cells for Rhodococcus equi. Activation of FcγRI can induce phagocytosis, antigen presentation, cytokine production, antibody-dependent cell mediated cytotoxicity, reactive oxygen species (ROS) production, and induction of autophagy machinery in macrophages. Here we observe enhanced macrophage bactericidal activity against intracellular R. equi concurrent with superoxide production and induction of autophagy. Following ligation of IFN-γ, TLR4 and FcγRI receptors, murine bone marrow-derived macrophages, equine pulmonary-alveolar macrophages and monocyte-derived macrophages were tested for superoxide production, induction of autophagy and bacterial load through multispectral imaging flow cytometry, western blot and confocal microscopy. Results show that ligation of these receptors alone or in combination enhanced superoxide production and induction of LC3II. Importantly, however, FcγRI activation with a synthetic homodimeric peptide resulted in a sustained effect leading to a profound reduction in bacterial load as compared with other receptor agonists. These data are consistent with the notion that production of superoxide enhances autophagy resulting in reduction of bacterial load in infected macrophages.