Phlebotomine sand flies are the biological vectors of Leishmania spp. and host complex microbial communities that may modulate insect physiology, ecological adaptation, and vector competence. In Colombia, where leishmaniasis remains a significant public health concern, characterization of sand fly-associated microbiota may provide insights into host–microbe–parasite interactions and identify candidates for microbiome-informed control strategies. Here, we characterized the bac-terial and fungal microbiomes of field-collected female sand flies from three municipalities in Antioquia, Colombia. Specimens were identified using morphological characters and confirmed by cytochrome C oxidase subunit I sequencing. Bacterial and fungal community composition was assessed by amplicon sequencing of the 16S rRNA gene and the internal transcribed spacer 2 (ITS2) region, respectively. To determine natural parasite infection status, all specimens were screened for Leishmania by nested PCR targeting the cyt b gene. Five sand fly species were de-tected, including the recognized vector species Nyssomyia olmeca bicolor, Lutzomyia gomezi, and Psychodopygus panamensis, and the non-vectors Pressatia dysponeta and Pr. camposi. No specimen tested positive for Leishmania. Across taxa, bacterial communities were dominated by members of the phyla Proteobacteria, Firmicutes, and Actinobacteria. In contrast, Bacteroidetes exhibited significantly higher relative abundance in vector species than in non-vectors, suggesting possible associations between bacterial community structure and vector status. Fungal communities were primarily composed of Basidiomycota and Ascomycota, and beta-diversity analyses indicated significant compositional differentiation among genera. Collectively, these data demonstrate that sand flies from endemic areas of Colombia harbor taxonomically distinct bacterial and fungal assemblages, with detectable differences between vector and non-vector species.