Classifying organisms into guilds is a good strategy to deal with taxonomic differences, and possibly to compare systems worldwide to address ecosystemic questions. Functional guilds have been used to classify organisms; however different classifications frequently are used for the same species, depending on each author. In order to standardize the guild classification for the Patos Lagoon Estuary, South Brazil, we evaluated its fish assemblage using historical dataset (1979 through 2016). Fish species were classified into guilds covering estuarine use, feeding mode and reproductive strategy. We found 140 Osteichthyes species for this estuary, in that most of the species are marine (58.5%), followed by freshwater (31.4%), estuarine (6.3%), and diadromous (3.8%) species. Concerning feeding mode, most species were classified as zoobenthivores, followed by piscivores and zooplanktivores, with some species presenting ontogenetic variation in their feeding habits. The average trophic level of this fish assemblage was estimated in three. Regarding reproduction, oviparity was the main reproductive mode, with pelagic eggs in the marine species, and settled eggs in the freshwater species. We provided an updated and unified list of Osteichthyes from Patos Lagoon Estuary, with all species classified in standardized guilds, assisting future studies that aim at analyzing estuarine assemblages in the Warm Temperate Southwestern Atlantic Province, as well as worldwide.