Social insects can establish nutritional, communication and defensive symbiotic associations with microorganisms, among which actinobacteria play important roles in these symbioses. Stingless bees are characterized by keeping their colonies free of diseases and parasites. These bees have evolved different protective strategies, but most are still unknown. Here we explore the potential of stingless bee Melipona scutellaris to associate with actinobacteria to suppress pathogenic microbes. Seven actinobacterial strains were isolated from foraging and nurse bees and identified through 16S rRNA sequencing. Three of these strains exhibited pronounced growth inhibition against the specialized pathogen of honey bees Paenibacillus larvae, the causative agent of American Foulbrood. Bioassay-guided HPLC fractionation, followed by NMR and mass spectrometry-based structure elucidation, revealed the presence of lobophorin (1–4) and anthracycline (5–15) families isolated from strains Streptomyces sp. ICBG1323 and Micromonospora sp. ICBG1321, all responsible for this activity. Our results suggest that M. scutellaris bees engage in defensive symbiosis with actinobacteria that produce antibacterial small molecules, helping to keep their colonies healthy.