Author summary According to the World Health Organization Leishmaniasis is the second parasitic disease that kills the most in the world; the first is malaria. Despite this, knowledge about theLeishmaniaparasite and its interaction with vertebrate hosts concerning the transmitting insect is still relatively fewer and fragmented. Studies on insects microbiota have great importance to obtain basic information. How a vector responds to the presence of different microorganisms and how they interact with various pathogens and may lead to the development of new strategies or tools that can be used to prevent or hinder the transmission of the protozoan by the vector insect. Considering the knowledge about the intestinal microbiota of sandflies, we aim to study the effect of bacterial isolates onLu.longipalpisinfection by different species ofLeishmania, and it believed that these bacteria might influence the development ofLeishmania, preventing, and hindering transmission, contributing to Leishmaniasis control strategies. The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere withLeishmaniadevelopment inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly inLutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions ofLu.longipalpis(Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus,Enterococcus,Erwinia,Enterobacter,Escherichia,Klebsiella,Lysinibacillus,Pseudocitrobacter,Providencia,Pseudomonas,Serratia,StaphylococcusandSolibacillus). Thein vitroandin vivoeffects of each one of the 13 native bacteria from theLu.longipalpiswere analyzed by co-cultivation with promastigotes ofL.i.chagasi,L.major,L.amazonensis, andL.braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated withLysinibacillus, all parasites ofL.infantum chagasiandL.amazonensisdied within 24 hours. In thein vivoco-infection ofL.chagasi,L.majorandL.amazonensiswith the generaLysinibacillus,PseudocitrobacterandSerratiait was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus,Serratia, andPseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence forLeishmania.