Bombus spp. are among the most important wild pollinators in natural and agricultural habitats. Bumblebees, like other pollinators, have suffered recent dramatic declines in their populations. This could be the result of changes in land use and pathogens and pathogens infections. In this study, we examined whether the diet and castes (queen and worker) influenced gut microbiota and the development of pathogen, under laboratory conditions. Ten fertilized queens of Bombus pauloensis and 10 of Bombus bellicosus were collected and carried out to the laboratory. They were caged and fed with two different diets ad libitum: monofloral bee bread (Eucalyptus grandis) or polyfloral bee bread. After the microcolonies were developed, at 5 days, worker of each group was sampled and their queens to evaluate the gut microbiota (16S rRNA gene sequencing) and the prevalence of Nosema ceranae, Nosema apis, Nosema bombi and Lotmaria passim (final time PCR). We observed that gut microbiota from both species of Bombus was mainly composed of Snodgrasella spp., Gilliamella spp., Bifidobacterium spp. and Lactobacillus spp. Diet pollen affected the gut microbiota of B. pauloensis and B. bellicosus. Monofloral pollen increased the richness of gut microbiota from both Bombus species, mainly increased the Gilliamella sp abundance. On the other hand, we found differences in the gut microbiota from different castes of B. bellicosus. Queens had more diverse gut microbiota than workers. The presence of pathogens evaluated was not detected in any samples. These results suggest that nutrition influences the abundance key components of gut microbiota in B. pauloensis and B. bellicosus while only gut microbiota in queen and worker of B. bellicosus were different. Those results contribute to the understanding of the influence of agriculture intensification and how Bombus spp. can adapt to different environmental conditions.