Black flies (Diptera: Simuliidae) are significant pests and vectors transmitting pathogens to humans and other animals. However, knowledge of species diversity as well as their role as pests and vectors in Laos is limited, despite the country having abundant suitable lotic habitats, that is, running water. In this study, we collected wild adult black fly specimens from Laos. Both morphology and genetic data supported that they were Simulium khelangense Takaoka, Srisuka and Saeung, and therefore, a new distribution record for this species. Screening of the blood protozoa from 106 females detected Leucocytozoon (n = 7) and Trypanosoma (n = 9). Identifications of Leucocytozoon based on cytochrome b sequences in MalAvi database indicated that 5 specimens had identical sequences to those of 4 lineages (GALLUS17 [n = 2], GALLUS35 [n = 1], GALLUS37 [n = 1], and GALLUS41 [n = 1]) of unidentified species (i.e., Leucocytozoon sp.).The remaining 2 had no identical sequences but were very similar (99%) to GALLUS34 and GALLUS41 lineages. Identifications of Trypanosoma based on the small subunit rRNA indicated that all were T. avium. The results presented here further support the role of S. khelangense as a potential vector of Leucocytozoon and Trypanosoma as well as being a possible pest of domestic avian species.