Ascidians are fast-growing sessile animals, frequently observed on artificial and natural substrata. Several introduced ascidians are known for monopolizing space in benthic communities, mainly in urbanized areas, where harbours act as gateways for exotic species. In the southeastern Brazil, the Arraial do Cabo Bay is the main point of coastal upwelling and one of the most visited localities during the summer due to beaches and dive sites. While the region of the botton of the bay is close to the city, being exposed to anthropogenic disturbances but warm waters, the area outside the bay is a pristine site, exposed to cold waters caused by the upwelling system. To understand how ascidian species are distributed through the temperature and anthropogenic gradients, we sampled ascidian specimens at 11 sites from three distinct regions: (1) inner bay, a harbor area exposed to intense ship and boat/platform traffic; (2) external bay, a pristine and warm area and (3) outside of the bay, a region exposed to coastal upwelling and cold water. We recorded 31 species of ascidians of which the Didemnidae and Styelidae families were the most speciose. Symplegma rubra and Phallusia nigra were the most common species. We found 22 species in the inner bay, of which nine were exclusive to this region (Styela canopus, Clavelina oblonga, Polyclinum constellatum, Polyclinum molle, Didemnum speciosum, Botrylloides giganteum, Ascidia curvata, Ascidia sydneiensis and Rhodosoma turcicum). In the external bay, we found 19 species, five exclusive to this region (Didemnum vanderhorsti, Didemnum galacteum, Eusynstyela tincta, Eusynstyela sp. and Botryllus sp.). Outside of the bay, three of the four species found (Cystodytes dellechiajei, Didemnum granulatum and Didemnum rodriguesi) were exclusive. Sixteen species were categorized as cryptogenic, seven as native and five as invasive to the Brazilian Coast. Four of the five invasive species were found only in the inner bay (R. turcicum, S. canopus, A. sydneiensis and A. curvata) close to man-made structures, while Styela plicata, an invasive ascidian with worldwide distribution, was also observed in the external bay. Seventeen of the 31 species were exclusive to one of the three regions, suggesting that anthropogenic impacts in the inner bay and the upwelling outside of the bay may be influencing the occurrence of different species, and increasing the local diversity of ascidians.