This paper discusses new findings relating to the first known diversity of trichomycete gut symbionts from Colombia. All species were associated with aquatic insects in the orders Diptera and Ephemeroptera collected from various localities in the tropical Eastern Ridge of Santander. Among the reported taxa, we describe two new species, Gauthieromyces colombiensis (Harpellales), phenotypically similar to the European G. microsporus, and Paramoebidium santanderensis (Amoebidiales) characterized by retaining the cystospore attached to the growing thallus after germination. Both taxa were found in the hindgut of mayfly nymphs. We also provide 17 new geographic records of trichomycetes for Colombia. Taxonomic and biogeographic implications of these records are discussed.