The applicability of two organic geochemical proxies (U37K' based on alkenones and TEX86H based on glycerol dialkyl glycerol tetraethers) for ocean temperatures was investigated on a regional scale in the SE Brazilian continental margin (Campos Basin) using 53 core-top sediments. In 71% of the samples for U37K' and 85% for TEX86H, the estimated annual mean sea surface temperature (SST) is comparable to the climatological annual mean data, with deviations within the calibration error. The surface TEX86H estimates reflects the surface mixed layer, while the depth-integrated (0–200 m) TEX86H estimates showed better agreement with temperatures at 100–200 m depth. In addition, the SST-U37K' results were more consistent (73% of the samples) using a winter calibration rather than an annual mean calibration. This seasonal effect might be ascribed to an increase in the abundance of haptophytes in the region during the winter. Exceptions to these general trends were observed for both proxies as a strong cold bias in reconstructed temperature in a few samples in the southern portion of the studied region, which may be related to (i) a predominant subsurface habitat of Thaumarchaeota combined with enhanced primary production and flux of particles during upwelling and (ii) alkenones being laterally transported from adjacent colder waters. Effects of river input of terrestrially sourced GDGTs on TEX86H are not evident. Overall, our findings show that U37K' and TEX86H are suitable temperature proxies for paleoclimate studies at a regional scale in the SE Brazilian continental margin, but on a local scale the effects of upwelling must be taken in consideration.