Several microorganisms can survive in harsh acid environments in geothermal springs at high temperatures across the Equatorial Andes Mountains. However, little is known about their physiological features and phylogenetic composition. Here we identify thermophilic microorganisms (bacteria, fungi, and microalgae) hosted in an almost unexplored geothermal spring (known as “Aguas Hediondas”). The phylogeny of the cultures was determined by analyzing physiological features and DNA sequences of PCR products for 16S rRNA, ITS, and 23S rRNA genes. Twenty pure cultures were isolated from the samples, including 17 for bacteria, one for cyanobacterium, one for eukaryotic microalgae, and one for fungus. Most bacterial strains were gram-positive, spore-forming, and bacilli (Bacillus). Cyanobacterium strain belonged to Chroococcidiopsis and the eukaryotic microalgae to Chlorophyta. The unique fungal strain isolated was closely related to T. duponti. Through our study, isolated thermophilic bacteria, microalgae and fungi from the “Aguas Hediondas” geothermal spring were characterized and identified. This study represents one of the first extensive molecular characterizations of extremophile microbes in the Tropical Equatorial Andes. Keywords. microbial diversity; DNA markers; extremophiles; phylogenetics