The rapid spread of antibiotic-resistance genes (ARGs) in Enterococcus faecalis (E. faecalis) poses a great challenge to human health and ecological and environmental safety. Therefore, it is important to control the spread of ARGs. In this study, we observed that the addition of 5 μg/mL docosahexaenoic acid (DHA) reduced the conjugative transfer of pCF10 plasmid by more than 95% in E. faecalis. DHA disturbed the pheromone transport by inhibiting the mRNA levels of the prgZ gene, causing the iCF10 pheromone to accumulate in the donor bacteria and bond to the PrgX receptor to form an inhibitory phase, which resulted in the down-regulation of the expression of genes related to conjugative transfer, inhibiting biofilm formation, reducing bacterial adhesion and thus inhibiting conjugative transfer. Collectively, DHA exhibited an admirable inhibitory effect on the transfer of ARGs in E. faecalis. This study provided a technical option to control the transfer of ARGs.