Highlights: • Fly larvae acquire microbial symbionts from their mothers and the fruit substrate. • Adult males-associated microorganisms also contribute to larval microbiota. • Larvae-associated yeasts can persist through fly life cycle and over generations. • Transmission rates vary greatly among microbial strains. Summary: Understanding the ecological and evolutionary dynamics of host-microbiota associations notably involves exploring how members of the microbiota assemble and whether they are transmitted along host generations. Here, we investigate the larval acquisition of facultative bacterial and yeast symbionts of Drosophila melanogaster and Drosophila suzukii in ecologically realistic setups. Fly mothers and fruit were major sources of symbionts. Microorganisms associated with adult males also contributed to larval microbiota, mostly in D. melanogaster. Yeasts acquired at the larval stage maintained through metamorphosis, adult life, and were transmitted to offspring. All these observations varied widely among microbial strains, suggesting they have different transmission strategies among fruits and insects. Our approach shows microbiota members of insects can be acquired from a diversity of sources and highlights the compound nature of microbiotas. Such microbial transmission events along generations should favor the evolution of mutualistic interactions and enable microbiota-mediated local adaptation of the insect host. + https://ars.els-cdn.com/content/image/1-s2.0-S2589004223017339-fx1.jpg