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The study aimed to discover quorum sensing (QS) inhibitors from marine sponge–derived actinomycetes and analyse its inhibitory activities against QS-mediated virulence factors in Pseudomonas aeruginosa. Seventy-two actinomycetes isolated from marine invertebrates collected from the western coast of India were screened against the QS indicator strain Chromobacterium violaceum CV12472. Methanol extracts of 12 actinomycetes showing inhibition of violacein production were accessed for downregulation of QS-mediated virulence factors like swarming, biofilm formation, pyocyanin, rhamnolipid and LasA production in Ps. aeruginosa ATCC 27853. The isolates NIO 10068, NIO 10058 and NIO 10090 exhibited very good anti-QS activity, with NIO 10068 being the most promising one. Mass spectrometric analysis of NIO 10068 methanol extract revealed the presence of cinnamic acid and linear dipeptides proline–glycine and N-amido-α-proline in the active extract. Detailed investigation suggested that although linear dipeptide Pro–Gly is to some extent responsible for the observed biological activity, cinnamic acid seems to be the main compound responsible for it. Marine-derived actinomycetes are a potential storehouse for QS inhibitors.This is the first report not only on marine sponge–associated Streptomyces for anti-QS in Ps. aeruginosa but also on cinnamic acid and proline-derived linear dipeptides proline–glycine as QS inhibitors. The results reveal that marine-derived actinomycetes may not only play a role in the defensive mechanism of their host but also lead to new molecules useful in the development of novel antivirulence drugs.