Aim: This study investigates the antimicrobial effects of fish oil-based formulas rich in omega-3 fatty acids (free fatty acids, ethyl esters or triacylglycerols), against cystic fibrosis (CF) pathogens (Burkholderia cenocepacia K56-2 and Pseudomonas aeruginosa PAO1), often resistant to multiple antibiotics. Methods and Results: The fish oils have shown antibacterial efficacy, although activity was highest for the one containing the fatty acid EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in their free form (MIC value is 1.87% v/v for both pathogens). To test whether the fish oils could have a therapeutic and prophylactic potential in vivo, we assessed its efficacy using a Galleria mellonella caterpillar model of infection. The treatment of infected larvae with a single dose (7 h post infection) enhances the survival of larvae, being more pronounced with the free fatty acid form (EPAX 6000 FA). Moreover, we observed that the prophylactic food provision of the fish oil EPAX 6000 FA during 12 days prior to bacterial infection extended the life of the infected larvae. Conclusion: The fish oils, particularly in the free fatty acid form, are active in killing Burkholderia and Ps. aeruginosa. Significance and Impact of the Study: The possibility of using fish oils for the treatment of bacterial infections in CF patients.