The well-studied, ancient and highly evolved mutualism between fungus-growing ants and their fungi has become a model system in the study of symbiosis1,5. Although it is thought at present to involve only two symbionts, associated with each other in near isolation from other organisms1,5, the fungal gardens of attine ants are in fact host to a specialized and virulent parasitic fungus of the genus Escovopsis (Ascomycotina)6. Because the ants and their fungi are mutually dependent, the maintenance of stable fungal monocultures in the presence of weeds or parasites is critical to the survival of both organisms. Here we describe a new, third mutualist in this symbiosis, a filamentous bacterium (actinomycete) of the genus Streptomyces that produces antibiotics specifically targeted to suppress the growth of the specialized garden-parasite Escovopsis. This third mutualist is associated with all species of fungus-growing ants studied, is carried upon regions of the ants' cuticle that are genus specific, is transmitted vertically (from parent to offspring colonies), and has the capacity to promote the growth of the fungal mutualist, indicating that the association of Streptomyces with attine ants is both highly evolved and of ancient origin.