<p>Marine biofouling remains a continuous problem in aquaculture and the maritime industry, which leads to the development of eco-sustainable antifouling agents. In the present study, ten marine bacteria were isolated from different marine habitats. These bacteria were subjected to bioassays for the production of secondary metabolites. The ethyl acetate extracts of these isolates were subjected to antibiofilm and antifouling studies against biofilm-producing <i>Pseudomonas</i> species. A combination of bioassays, including crystal violet staining for biofilm biomass estimation, phenol-sulphuric acid assay for extracellular polymeric substance (EPS) estimation, ATP bioluminescence assay for metabolic viability, and inhibition of bacteria and diatoms, was performed. Thin-Layer Chromatography (TLC) and Gas C hromatography-Mass Spectrometry (GC-MS) studies revealed the presence of a variety of secondary metabolites in the extracts. The Rf value ranged from 0.46 to 0.74. The presence of phthalate derivatives, long-chain hydrocarbons, and aliphatic alcohols was detected by comparing the GC-MS library. Among the extracts, MB2 and MB23 were found to possess the highest antibiofilm activity by inhibiting 80–86% biofilm formation, with a range of 0.5–1.0&#xa0;µg/mL. These extracts were found to possess moderate toxicity by <i>Artemia salina</i> bioassay (LC₅₀119-156&#xa0;µg/mL), which suggests their suitability as environmentally friendly antifouling compounds. This study demonstrates the potential of marine bacterial extracts as promising candidates for the discovery of new antifouling compounds.</p> Graphical Abstract <p></p>