ABSTRACT Introduced species are one of the biggest threats to aquatic ecosystems. Rainbow trout ( Oncorhynchus mykiss , Salmonidae) is considered one of the most dangerous introduced predatory fish species, as they often put native species at risk of extinction. This study evaluated the effects of rainbow trout introduction on Andean stream food webs. We tested the hypothesis that the presence of rainbow trout changes Andean stream food webs by changing the diet of other carnivorous species and the energy source supporting native fish species. We sampled streams with and without rainbow trout and with different vegetation cover (i.e., pastures and crops versus forest) and combined data from stomach contents and stable isotopes. We analyed stomach contents from 231 specimens of two native catfish species and 116 rainbow trout specimens. Our results demonstrate that aquatic insects are essential in Andean stream food webs, where collector gatherers and scrapers were the most consumed and assimilated by catfish and rainbow trout species. Leaf litter was predicted to be the primary energy source in streams with forest cover. In contrast, periphyton contributed the most in streams with pastures and crops. We conclude that the presence of trout coupled with use/land cover (i.e., vegetation cover) exerts a substantial effect on Andean stream food webs. Protection of riparian forests of the Andean streams of Colombia is needed to guarantee the stability of aquatic food webs and thus help native species coexist with established non‐native species introduced decades ago.