The diversity and biotechnological potentialities of bacterial isolates from brines of three Antarctic lakes of the Northern Victoria Land (namely Boulder Clay and Tarn Flat areas) were first explored. Cultivable bacterial communities were analysed mainly in terms of bacterial response to contaminants (i.e., antibiotics and heavy metals) and oxidation of contaminants (i.e., aliphatic and aromatic hydrocarbons and polychlorobiphenyls). Moreover, the biosynthesis of biomolecules (antibiotics, extracellular polymeric substances and enzymes) with applications for human health and environmental protection was assayed. A total of 74 and 141 isolates were retrieved from Boulder Clay and Tarn Flat brines, respectively. Based on 16S rRNA gene sequence similarities, bacterial isolates represented three phyla, namely Proteobacteria (i.e., Gamma- and Alphaproteobacteria), Bacteroidetes and Actinobacteria, with differences encountered among brines. At genus level, Rhodobacter, Pseudomonas, Psychrobacter and Leifsonia members were dominant. Results obtained from this study on the physiological and enzymatic features of cold-adapted isolates from Antarctic lake brines provide interesting prospects for possible applications in the biotechnological field through future targeted surveys. Finally, findings on contaminant occurrence and bacterial response suggest that bacteria might be used as bioindicators for tracking human footprints in these remote polar areas.