Considering the insufficient scientific knowledge of diatoms attached to plants in wetlands, this study aims to present their floristic composition in Arroio Pseudônimo and also images of species that represent first records for the State of Rio Grande do Sul, Brazil. Twelve samples from the fern Acrostichum danaeifolium fronds were collected, over four seasons (from May 2011 to January 2012). Acrostichum danaeifolium has a limited distribution in Arroio Pseudônimo (Lagoa Pequena), Pelotas. Epiphytic material was removed and permanent slides were prepared for observation and identification. Physical (temperature, depth, water transparency and flow) and chemical (pH, electrical conductivity, phosphorus and nitrogen) variables of water were measured. The diatom flora showed high species richness (157); Nitzschia displayed the highest number of taxa (22). The community was composed of taxa characteristic of fresh water (26%), estuarine (50%) and marine (14%), from classes Bacillariophyceae (75%), Coscinodiscophyceae (17%) and Fragillariophyceae (8%). The greatest richness occurred in summer (99 species) and autumn (90), compared to spring (46) and winter (29). Twenty-three species are recorded for the first time to Rio Grande do Sul.