Description: Porifera dominate vast areas of the Antarctic shelves and are successfully colonized by bacteria. Quorum sensing (QS) is a cell-to-cell communication system based on bacterial population density that, enabling the coordination of group-based behaviour, plays a critical role in the successful colonization of higher organisms, also driving the formation of biofilm for adhesion to surfaces. In this study, the production of N -Acyl homoserine lactones (AHLs), signal molecules involved in the QS mechanism, was examined for 211 Antarctic sponge-associated Gram-negative bacteria. AHL production was screened by using three different AHL biodetection systems, i.e. Agrobacterium tumefaciens pZLR4, Chromobacterium violaceum CV026 and Pseudomonas putida pKR-C12 with optimal sensitivity to moderate-chain (C8–C12), short-chain (C4–C8) and long-chain (≥ C14) AHLs, respectively. 57.8% of tested isolates activated at least one of the monitor systems used and belonged mainly to bacterial genera that are known to be involved in surface colonization by biofilm production. A thin-layer chromatographic assay based on the A. tumefaciens reporter system was utilized to determine the AHL profiles of five selected positive isolates. Visible spots on thin-layer chromatography (TLC) plates were produced by Roseobacter sp. TB60 and Psychrobacter sp. TB67 (both from the sponge, Anoxycalyx joubini ). The former probably produced N -(3-oxohexanoyl)-L-homoserine lactone (similar to the standard 3-oxo-C6-HSL), whereas the isolate TB67 produced molecules that were similar to the standard N -butanoyl-homoserine lactone (C4-HSL). The obtained results demonstrated that AHL-based signalling may play a key role in sponge–bacteria interactions also in the Antarctic environment.