The objective was to explore levels of persistence and stability of a Neotropical fish assemblage in a typical Serra do Mar stream (Southeast Brazil) over 1994–2008 with 90 quantitative samples in total. All along the stream gradient, the fish assemblage was composed of small-sized, short living species including benthic and water column species. Only four species occurred at the uppermost site (i.e., S1) whereas these four species plus eight species occurred at all other sites. In regards to species presence-absence, all species were temporally persistent at all sites over the study years. Overall, all species were abundant across scales of space and time with only three out of 14 species showing consistently low densities along the stream across years. Nevertheless, as indicated by the temporal variations in the Coefficients of Variation (CV%), the fish assemblages were highly unstable at all sites over time. At all sites the CV% varied widely and increased significantly over the years. No consistent density-dependence was elucidated nor any significant correlation among environmental variables and assemblage attributes were detected. The only consistent relationship elucidated relates the CV% of assemblage variability over time and the CV% of the ratio between stream discharge in the two major seasons, May and November.