Especially added on many industrial and domestic products as flame retardants (FRs), polybrominated diphenyl ethers (PBDEs) are among the chemicals of high environmental concern because of their potential harmfulness for environmental and human health. Seafood consumption is considered the main source of PBDEs and their methoxylated congeners (MeO-BDEs) for humans. The present study aims to investigate the seasonal occurrence of six PBDEs and eight MeO-BDEs congeners using Douro river biota (different trophic levels) as sentinels, as well as to evaluate the human exposure risk to PBDEs through seafood consumption. Biota samples (n = 273) were collected from one of the most important Portuguese estuaries in the north-western coast of Portugal at four different seasons (2019–2020). The analyses were performed by an environmental-friendly extraction procedure followed by Gas Chromatography coupled to a triple quadrupole detector (GC–MS/MS). PBDEs were detected in all seafood samples analysed, with means ranging from 0.02 ng g−1 ww (flounder in autumn) to 3.75 ng g−1 ww (mussel in winter). Levels of lower-brominated PBDE congeners were significantly higher than higher-brominated ones in all seasons (p < 0.01). MeO-BDEs ranged from 0.001 ng g−1 ww (grey mullet in summer) to 5.66 ng g−1 ww (green crab in spring). Crabs and mussels presented the highest means of PBDEs and MeO-BDEs. Regarding the health risk assessment of the studied PBDE congeners (47, 99, and 153), consumption of Douro river fish is not a case of concern for consumers. Display omitted •PBDEs and their metabolites were found in Douro river biota.•Higher levels of MeO-BDEs were found in both invertebrate and macroalgae species.•PBDEs and MeO-BDEs levels varied among seasons.•Low risk through fish consumption according to the MOE threshold for PBDEs.