Little is known on the origin and maintenance of symbionts associated with Drosophila larvae in natura , which restricts the understanding of Drosophila -extracellular microorganism symbiosis in the light of evolution. Here, we studied the origin and maintenance of symbionts of Drosophila larvae under ecologically realistic conditions, to our knowledge for the first time, using yeast and bacterial isolates and two Drosophila species: the model organism D. melanogaster and the invasive pest D. suzukii . We discovered that Drosophila females and males both transmit yeast and bacteria symbionts to larvae. In addition, several symbiotic yeasts initially associated with larvae were conserved throughout host life cycle and transmitted to offspring. Our results suggest that stable associations of Drosophila flies with bacteria and yeasts may exist in natura and constitute a step forward in the understanding of wild Drosophila -microorganism symbioses.