Emerging evidence suggests that Omega-3 polyunsaturated fatty acids (PUFAs) are essential structural and functional nutrients that significantly influence the composition and function of the gut microbiota, a key mediator of host health. Although evidence suggests that increasing tissue Omega-3 levels through dietary intervention may optimize gut microbiota-host interaction through modulation of composition, metabolite production, and the intestinal mucus barrier, some studies have reported inconsistent findings regarding these protective effects. Studies indicate that a high Omega-6/Omega-3 ratio appears to attenuate the beneficial effects of Omega-3 supplementation on microbial diversity and abundance, while a balanced ratio fosters a more favorable microbiome profile. This review comprehensively highlights the potential effects of differential endogenous Omega-6/Omega-3 ratios on the gut microbiota-modulating capacity of Omega-3 PUFAs, which should be incorporated as a mandatory monitoring indicator in future clinical investigations. These insights provide a new direction for further optimizing the clinical application of Omega-3 PUFAs in chronic disease prevention and treatment.