Propolis is a resinous material produced by honeybees from different plant sources and used in the hive as a building material and to protect the colony from parasites and pathogens. Despite its antimicrobial properties, recent studies showed that propolis hosts diverse microbial strains, some with great antimicrobial potential. In this study, the first description of the bacterial community of propolis produced by the gentle Africanized honeybee was reported. Propolis was sampled from hives of two different geographic areas of Puerto Rico (PR, USA), and the associated microbiota investigated by both cultivation and metataxonomic approaches. Metabarcoding analysis showed appreciable bacterial diversity in both areas and statistically significant dissimilarity in the taxa composition of the two areas, probably due to the different climatic conditions. Both metabarcoding and cultivation data revealed the presence of taxa already detected in other hive components and compatible with the bee’s foraging environment. Isolated bacteria and propolis extracts showed antimicrobial activity against Gram-positive and Gram-negative bacterial tester strains. These results support the hypothesis that the propolis microbiota could contribute to propolis’ antimicrobial properties.