Fish oil, enriched in omega-3 polyunsaturated fatty acids (n-3 PUFA), is widely used as a dietary or nutritional supplement with numerous benefits, including as an anti-inflammatory particularly linked to atherosclerosis. While n-3 PUFA have been suggested to be able to improve oral health through a reduction in inflammation through elevations in these fatty acids in serum and cellular membranes, information is lacking for the possibility that these fatty acids could directly impact the survival and growth of the oral bacteria that trigger the chronic inflammatory responses. The n-3 fatty acids, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and alpha-linolenic acid (ALA), and their fatty acid ethyl esters, ALAEE, EPAEE, DHAEE were analysed for antibacterial activity against oral pathogens. This study demonstrated a novel bioactivity of the three major n-3 PUFA, EPA, DHA, and ALA, and their ester derivatives. Our experimental data indicated that n-3 PUFA and their ester derivatives exhibited strong antibacterial activity against various oral pathogens, including Streptococcus mutans, Candida albicans, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis. This study suggested that n-3 PUFA could have a positive therapeutic effect for improving oral health via their antibacterial activities, besides their anti-inflammatory effects.