Some species of mastiff bats, Molossus Geoffroy, 1805, inhabit human shelters such as houses and barns. Among them, the Pallas’s mastiff bat, Molossus molossus Pallas, 1766, is the most common species in South America. There are a few studies on this bat in Colombia, mostly on colony size, diet, ectoparasite records, and activity patterns in the Andean and Caribbean regions. Here, we provide information on the prevalence of chiggerfleas, Hectopsylla pulex (Haller, 1880), on M. molossus, along with molecular data on the flea, and its distribution in Colombia. In addition, we describe the size and sex ratio of the infested bat colony, located in the central Andes of Colombia. The bat colony was represented by ca. 45 individuals, of which 33 were captured. The colony had more females (25 individuals) than males (8 individuals). A total of four Pallas’s mastiff bats had chiggerfleas, H. pulex, most of which were attached to the bat’s faces and ears. The composition of the colony (sex ratio) and the observed activity times match those reported for other colonies of the species in Colombia. The cytochrome oxidase subunit 1, and the 12S rRNA mitochondrial gene obtained from H. pulex represent the second and first available sequences for the species, respectively. The level of infestation of individuals in the colony was low, similar to that observed in other South American countries, such as Brazil. Finally, the new locality is the seventh confirmed and the highest elevational record of H. pulex in Colombia.