Microplastic pollution is a widespread environmental concern. Like other anthropogenic pollutants, microplastics can reach aquatic ecosystems through rivers and interact with the aquatic biota. For instance, Lake Titicaca (between Bolivia and Peru), one of the great ancient lakes in South America (3,809 m a.s.l.), shows a pollution problem, particularly in the southern shallow basin (Lago Menor) in Bolivia. Nevertheless, our knowledge of the presence of microplastics and their interaction with the biota of Lake Titicaca is limited. Therefore, this study evaluated the presence of microplastics in the stomach content of the four fish species targeted by local fisheries in Lago Menor of Lake Titicaca (Orestias luteus, Orestias agassizii, Trichomycterus dispar, and Odonthestes bonariensis; N = 1,283), and looked for relationships with trophic guilds or fishing areas. Additionally, surface water was analyzed to evaluate the presence of microplastics in the water. The evaluation of microplastics was carried out by visual observations. We observed that the frequency of microplastic ingestion was low in all species (<5%). Conversely, microplastic was present in the water, with the highest quantity at the southern part of Lago Menor (103 ± 20 particles per L), without differences in the microplastic number between sites. Most microplastics counted in stomach contents were fibers, whereas water samples mainly contained fragments. Our results point to microplastic pollution in Lago Menor of Lake Titicaca. However, we could not determine the pollution rate due to considerable methodological limitations. Further research will be needed to robustly detect microplastics in Lake Titicaca and their impact on the fish species in the lake.