Biofouling is a phenomenon that describes the fouling organisms attached to man-made surfaces immersed in water over a period of time. It has emerged as a chronic problem to the oceanic industries, especially the shipping and aquaculture fields. The metal-containing coatings that have been used for many years to prevent and destroy biofouling are damaging to the ocean and many organisms. Therefore, this calls for the critical need of natural product-based antifoulants as a substitute for its toxic counterparts. In this study, the antibacterial and antibiofilm activities of the bioactive compounds of Pseudoalteromonas sp. IBRL PD4.8 have been investigated against selected fouling bacteria. The crude extract has shown strong antibacterial activity against five fouling bacteria, with inhibition zones ranging from 9.8 to 13.7 mm and minimal inhibitory concentrations of 0.13 to 8.0 mg/ml. Meanwhile, the antibiofilm study has indicated that the extract has attenuated the initial and pre-formed biofilms of Vibrio alginolyticus FB3 by 45.37 ± 4.88% and 29.85 ± 2.56%, respectively. Moreover, micrographs from light and scanning electron microscope have revealed extensive structural damages on the treated biofilms. The active fraction was fractionated with chromatographic methods and liquid chromatography-mass spectroscopy analyses has further disclosed the presence of a polyunsaturated fatty acid 4,7,10,13-hexadecatetraenoic acid (C16H24O2). Therefore, this compound was suggested as a potential bioactive compound contributing to the antibacterial property. In conclusion, Pseudoalteromonas sp. IBRL PD4.8 is a promising source as a natural antifouling agent that can suppress the growth of five fouling bacteria and biofilms of V. alginolyticus FB3.