Background The increment of resistant strains to commonly used antibiotics in clinical practices places in evidence the urgent need to search for new compounds with antibacterial activity. The adaptations that Antarctic microorganisms have developed, due to the extreme environment that they inhabit, promote them as a potential new source of active compounds for the control of microorganisms causing infections associated with health care. The aim of this study was to evaluate the antibacterial activity of an ethanol extract of the Antarctic bacterium Janthinobacterium sp., strain SMN 33.6, against nosocomial multi-resistant Gram-negative bacteria. Results Inhibitory activity against human Gram-negative bacterial pathogens, with concentrations that varied between 0.5 and 16 µg ml- 1, was demonstrated. Conclusions The ethanolic extract of Janthinobacterium sp. SMN 33.6 possesses antibacterial activity against a chromosomal AmpC beta-lactamase-producing strain of Serratia marcescens, an extended-spectrum beta-lactamase-producing Escherichia coli and also against carbapenemase-producing strains of Acinetobacter baumannii and Pseudomonas aeruginosa. This becomes a potential and interesting biotechnological tool for the control of bacteria with multi-resistance to commonly used antibiotics.