Environmental factors, size-related isotopic changes of the most abundant species and isotopic niche overlap were investigated using stable isotopes in order to evaluate spatial changes of fish trophic guilds in the Araruama Lagoon. Based on 440 muscle samples, 17 fish species were grouped into five trophic guilds. Mean salinity was above 40 at both sites sampled and a significant spatial difference was observed. The highest δ13C mean value was observed for an omnivorous species, whereas the lowest carbon signatures were found for the three fish species belonging to the planktivorous guild. Analysis of the carbon signature of fish species in lower trophic levels showed influence of salinity variation, whilst size appeared to play a role for others. A narrow δ15N difference was observed, but the piscivorous fish species showed the highest δ15N values. The Standard Ellipses Analysis (SEA) detected spatial differences and varying degrees of isotopic niche overlap among trophic guilds, but the percentages of most overlaps (<60%) suggest that, to some extent, the guilds had a unique isotopic niche space. These results are in agreement with data previously reported for the Araruama Lagoon, that found the same prey items with varying relative importance among the most abundant species. Further studies are necessary to understand how the interaction between salinity and other factors, such as migration patterns, changes in prey availability, changes in contribution of primary sources and changes in baseline isotopic signatures could affect the stable isotope signatures shown here.