Popular as aquarium fish, armored catfishes from South America (Pterygoplichthys spp.) have been introduced and become invasive in tropical and subtropical regions worldwide. These ecosystem engineers can deplete basal resources (e.g., periphyton and detritus), with potential negative effects for native fauna. We studied the trophic ecology of fishes in the Usumacinta River Basin, Guatemala, where Pterygoplichthys is now widespread and locally abundant. We analyzed stable isotopes (δ13 C, δ15 N) in fish tissues and basal resources to assess the potential impact of Pterygoplichthys on the trophic ecology of six co-occurring native fishes that feed at a similar trophic level (Astyanax aeneus, Dorosoma petenense, Thorichthys pasionis, Oscura heterospila, Poecilia mexicana, and Gambusia sexradiata). The study was conducted during the dry season in the La Pasion (LPR; high invasion) and San Pedro (SPR; low invasion) rivers. We compared isotopic spaces occupied by native fish and Pterygoplichthys, estimated isotopic overlap, and evaluated the trophic displacement of native species. We also evaluated the relationships of environmental factors, including the relative biomass of the invasive catfish, with δ13 C and δ15 N signatures. Except for P. mexicana, native species had lower isotopic overlap with the catfish in LPR. Native fish isotopic spaces were compressed and shifted toward higher trophic positions in LPR relative to SPR. Benthic food resources were important for Pterygoplichthys in both rivers, and water-column resources had greater relative importance (RI) for native species in LPR. Native fish δ13 C was significantly associated with Pterygoplichthys biomass, conductivity, and water flow velocity; and water depth and sedimentation had a significant association with native fish δ15 N. Findings provide evidence that invasive Pterygoplichthys, along with environmental factors, impact the trophic ecology of native fish in the Usumacinta Basin. Additional field research conducted over longer time periods and mesocosm experiments that account for fish assemblage and environmental variation could elucidate Pterygoplichthys impacts via food resource depletion or habitat alteration.