Propolis has been the subject of several recent studies, with the aim of elucidating its biological and pharmacological properties. Propolis has a well-known antimicrobial activity as well as antioxidant, antitumoral, antiinflammatory, and regenerative properties, but literature about its effects on the immune response is scarce. The goal of this work was to evaluate the propolis effect on macrophage activation by oxygen (H2O2) and nitrogen (NO) metabolite determination. Propolis was produced by africanized honeybees and hydroalcoholic solutions were prepared at different concentrations. Peritoneal macrophages were obtained from male BALB/c mice and culture cells were stimulated in vitro with propolis or interferon-gamma (IFN-gamma). In the in vivo assay, the animals were sacrificed after propolis treatment and cells were stimulated with IFN-gamma. We also investigated the co-stimulant action of propolis associated with IFN-gamma on macrophages. The results show that propolis induces a discreet elevation in H2O2 release and a mild inhibition of NO generation, depending on concentration. Propolis had no co-stimulant activity, diminishing IFN-gamma action on H2O2 and NO production. Data suggest that propolis acts on host non-specific immunity by macrophage activation.