The populations of freshwater mussels belonging to the family Unionidae have been facing drastic changes in terms of diversity and numbers caused by constant aggressions on their natural habitat. Nevertheless, bivalves are capable of developing strategies of defense to overcome potential aggressors. The present work aimed to assess the potential antibacterial capacity of different species of unionid mussels from the north of Portugal. For this purpose, circulating cells (hemocytes), fluids, and mucus were obtained by nonlethal methods from the species Anodonta anatina (Linnaeus), Anodonta cygnea (Linnaeus), Potomida littoralis (Cuvier), and Unio delphinus (Spengler), and tested against bacterial reference strains and multidrug-resistant isolates. The cellular fraction of A. anatina, A. cygnea, and P. littoralis showed antibacterial activity, detected by the agar disc diffusion method, against Bacillus subtilis ATCC 6683, Pseudomonas aeruginosa ATCC 27853 and Acinetobacter baumannii ATCC 19606. Circulating cells from P. littoralis and A. anatina also inhibited Listeria monocytogenes ATCC 19111 and A. cygnea has also inhibited a multidrug-resistant isolate of Pseudomonas putida. The plasma of all mentioned freshwater mussels, used directly or diluted, showed great ability to hamper or inhibit the biofilm formation of Staphylococcus aureus ATCC 25923, P. aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. Anodonta cygnea hampered the biofilm formation by Enterococcus faecalis ATCC 29212 as well. Overall, these results showed that not only cells play a relevant role in the immune system of these species but also the plasma, which likely contains antibiofilm substances. Anodonta cygnea stood out by presenting the best antibacterial inhibition potential.