Occurrence of tetrodotoxin (TTX), a potent natural neurotoxin, in marine organisms and seafood from the European Union has been of increasing concern due to its relatively recent detection in bivalve mollusks and gastropods. Following a request of the European Food Safety Authority (EFSA) to EU Member States to obtain more data on TTX occurrence, this study collected 117 samples of bivalve mollusks, including mussels (Mytilus galloprovincialis), oysters (Crassostrea gigas) and clams (Ruditapes philippinarum and Donax spp.), from the South and Southwest Portuguese coast between May and October 2018, for TTX determination and microbiological analysis. The analyses carried out by hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) did not detect TTX in any sample, and microbiological analysis did not reveal high concentrations of Vibrio spp., which has been linked to the presence of TTX. Although preliminary, results from this study, the first investigating the presence of TTX in bivalve mollusks from the Portuguese coast, suggest that TTX may not represent a risk for human consumption of bivalve mollusks.