Abstract The mutualistic interaction specificity between attine ants and antibiotic-producing Actinobacteria has been controversial because Pseudonocardia strains cannot always be isolated from worker cuticles across attine ant species, while other actinobacteria can apparently replace Pseudonocardia and also inhibit growth of Escovopsis mycopathogens. Here we report that across field samples of Panamanian species: (i) Cuticular Pseudonocardia were largely restricted to species in the crown of the attine phylogeny and their appearance likely coincided with the first attines colonizing Central/North America. (ii) The phylogenetically basal attines almost always had cuticular associations with other Actinobacteria than Pseudonocardia. (iii) The sub-cuticular glands nourishing cuticular bacteria appear to be homologous throughout the phylogeny, consistent with an ancient general attine-Actinobacteria association. (iv) The basal attine species investigated always had Pseudonocardia as gut symbionts while Pseudonocardia presence appeared mutually exclusive between cuticular and gut microbiomes. (v) Gut-associated Pseudonocardia were phylogenetically ancestral while cuticular symbionts formed a derived crown group within the Pseudonocardia phylogeny. We further show that laboratory colonies often secondarily acquire cuticular Actinobacteria that they do not associate with in the field, suggesting that many previous studies were uninformative for questions of co-adaptation in the wild. An exhaustive literature survey showed that published studies concur with our present results, provided that they analyzed field colonies and that Actinobacteria were specifically isolated from worker cuticles shortly after field collection. Our results offer several testable hypotheses for a better overall understanding of attine-Pseudonocardia interaction dynamics and putative coevolution throughout the Americas.