As honey bee populations decline, interest in pathogenic and mutualistic relationships between bees and microorganisms has increased. Honey bees and bumble bees appear to have a simple intestinal bacterial fauna that includes acidophilic bacteria. Here, we explore the hypothesis that sweat bees can acquire acidophilic bacteria from the environment. To quantify bacterial communities associated with two species of North American and one species of Neotropical sweat bees, we conducted 16S rDNA amplicon 454 pyrosequencing of bacteria associated with the bees, their brood cells and their nests. Lactobacillus spp. were the most abundant bacteria in many, but not all, of the samples. To determine whether bee-associated lactobacilli can also be found in the environment, we reconstructed the phylogenetic relationships of the genus Lactobacillus. Previously described groups that associate with Bombus and Apis appeared relatively specific to these genera. Close relatives of several bacteria that have been isolated from flowers, however, were isolated from bees. Additionally, all three sweat bee species associated with lactobacilli related to flower-associated lactobacilli. These data suggest that there may be at least two different means by which bees acquire putative probiotics. Some lactobacilli appear specific to corbiculate apids, possibly because they are largely maternally inherited (vertically transmitted). Other lactobacilli, however, may be regularly acquired from environmental sources such as flowers. Sweat bee–associated lactobacilli were found to be abundant in the pollen and frass inside the nests of halictids, suggesting that they could play a role in suppressing the growth of moulds and other spoilage organisms.