To combat disease, most fungus-growing ants (Attini) use antibiotics from mutualistic bacteria (Pseudonocardia) that are cultured on the ants' exoskeletons and chemical cocktails from exocrine glands, especially the metapleural glands (MG). Previous work has hypothesized that (i) Pseudonocardia antibiotics are narrow-spectrum and control a fungus (Escovopsis) that parasitizes the ants' fungal symbiont, and (ii) MG secretions have broad-spectrum activity and protect ants and brood. We assessed the relative importance of these lines of defence, and their activity spectra, by scoring abundance of visible Pseudonocardia for nine species from five genera and measuring rates of MG grooming after challenging ants with disease agents of differing virulence. Atta and Sericomyrmex have lost or greatly reduced the abundance of visible bacteria. When challenged with diverse disease agents, including Escovopsis, they significantly increased MG grooming rates and expanded the range of targets. By contrast, species of Acromyrmex and Trachymyrmex maintain abundant Pseudonocardia. When challenged, these species had lower MG grooming rates, targeted primarily to brood. More elaborate MG defences and reduced reliance on mutualistic Pseudonocardia are correlated with larger colony size among attine genera, raising questions about the efficacy of managing disease in large societies with chemical cocktails versus bacterial antimicrobial metabolites.