Abstract This research aimed to study the composition of epilithic diatom flora found in samples collected in Cali River hydrographical basin, Colombia. Quarterly excursions (March, June, September and December of 2012 and March of 2013) were performed in six sampling sites to collect samples for identification of diatom organisms (Class Bacillariophyceae). The results indicated the occurrence of 82 taxa distributed in 26 families and 38 genera. 32 of these are new occurrences in Colombia. The most representative families were Gomphonemataceae, Naviculaceae and Bacillariaceae. The genera richest in species were Navicula, Gomphonema and Nitzschia. Eight species occurred in all sampling sites: Achnanthidium minutissimum, Cocconeis lineata, Cymbella affinis, Gomphonema pumilum var. rigidum, Melosira varians, Navicula symmetrica, Reimeria sinuata and Rhoicosphenia abbreviata. The results indicated 13 species considered dominant and characterize the flora of epilithic diatoms in Cali River hydrographical basin, Colombia.