Mixed infections by different Trypanosoma species or genotypes are a common and puzzling phenomenon. Therefore, it is critical to refine the diagnostic techniques and to understand to what extent these methods detect trypanosomes. We aimed to develop an accessible strategy to enhance the sensitivity of the hemoculture, as well as to understand the limitations of the hemoculture and the blood clot as a source of parasitic DNA.We investigated trypanosomatid infections in 472 bats by molecular characterization (18S rDNA gene) of the DNA obtained from the blood clot and, innovatively, from three hemoculture sample types: the amplified flagellates (“isolate”), the pellet of the culture harvested in its very initial growth stage (“first aliquot”), and the pellet of non-grown cultures with failure of amplification (“sediment”). We compared (a) the characterization of the flagellates obtained by first aliquots and isolates; and (b) the performance of the hemoculture and blood clot for trypanosomatid detection. We observed: (i) a putative new species of Bodo in Artibeus lituratus; (ii) the potential of Trypanosoma cruzi selection in the hemoculture; (iii) that the first aliquots and sediments overcome the selective pressure of the hemoculture; and (iv) that the blood clot technique performs better than the hemoculture. However, combining these methods enhances the detection of single and mixed infections.