This work provides information about the fish assemblage structure along the estuarine gradient of Baía da Babitonga, south Brazil. The seasonal and spatial dynamics of fish and their relationship with physical–chemical variables were investigated. A total of 70,085 fish of 70 taxa were collected. Late larva and early juveniles of Engraulidae, Eucinostomus spp. and Mugil spp. dominated in abundance, representing 62% of all fish captured. Permutational multivariate analysis of variance identified distinct fish assemblages within the bay and during the year. The marine straggler species Harengula clupeola, Oligoplites saliens and Trachinotus carolinus and the estuarine migrant Anchoa tricolor were characteristic of the outer-most bay area, while the estuarine resident and migrant species Atherinella brasiliensis, Anchoa januaria, Sphoeroides greeleyi and Citharichthys spilopterus, and the marine migrant Diapterus rhombeus were characteristic of the inner portion of the estuary. The seasonal changes in community structure observed were mainly related to the greater abundance of T. carolinus in the warm/wet season, Micropogonias furnieri in the transition season and Oligoplites saliens in the cold/dry season. Depth, followed by salinity, explained the greater part of the variability in the abundance of dominant species and was found to be important in shaping the assemblages. Nevertheless, the amount of variation unexplained by the measured abiotic variables was relatively high (73%), suggesting the effect of additional regulatory factors. Many fish species use the shallow waters of the bay in transitory or permanent ways, and knowledge about their relationship with the environment is necessary for the success of conservation strategies for this ecosystem.