An entomological survey at rural and cavernicolous localities in four provinces in southern Thailand provided 155 blood-fed females of sand flies (Diptera: Psychodidae) that were identified based on morphological characters as Idiophlebotomus asperulus (n = 19), Phlebotomus stantoni (n = 4), P. argentipes (n = 3), Sergentomyia anodontis (n = 20), S. barraudi (n = 9), S. hamidi (n = 23), S. hodgsoni (n = 4), S. hodgsoni hodgsoni (n = 32), S. indica (n = 5), S. iyengari (n = 2), S. khawi (n = 17), S. silvatica (n = 11) and Sergentomyia sp. (n = 6). The dominant species in this study was S. hodgsoni hodgsoni, which was collected specifically in a Buddha cave. Screening for DNA of parasitic protozoans revealed eight specimens (5.16%) of four species (S. barraudi, S. indica, S. khawi and Id. asperulus) positive for Trypanosoma sp., while no Leishmania spp. DNA was detected. Blood meals of engorged females were identified by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) assay on a fragment of cytochrome b (cyt b) gene with a success rate 36%, humans, dogs, and rats being determined as sources of blood. Bloodmeal analysis of two Trypanopsoma-positive females (S. barraudi and Sergentomyia sp.) identified blood from dogs and humans, respectively. Our findings indicate that S. barraudi, S. indica, S. khawi and Id. asperulus may be incriminated in circulation of detected Trypanosoma spp.