Development of new bacterial biofilm inhibitors as antibacterial synergists is an effective strategy to solve the resistance of <i>Pseudomonas aeruginosa</i>. In this paper, a series of 3-hydroxy-pyridin-4(1<i>H</i>)-ones were synthesized and evaluated, and the hit compound (<b>20p</b>) was identified with the effects of inhibiting the production of pyocyanin (IC<sub>50</sub> = 8.6 μM) and biofilm formation (IC<sub>50</sub> = 4.5 μM). Mechanistic studies confirmed that <b>20p</b> inhibits the formation of bacterial biofilm by inhibiting the expression of <i>pqsA</i>, blocking <i>pqs</i> quorum sensing system quinolone biosynthesis. Moreover, we systematically investigated the bactericidal effects of combining currently approved antibiotics for CF including tobramycin, ciprofloxacin, and colistin E with <b>20p</b>, which showed obvious antibacterial synergy to overcome antibiotics resistance in multidrug-resistant <i>P. aeruginosa</i> biofilms. The result indicates that compound <b>20p</b> may be used in the future as a potentially novel antibacterial synergist candidate for the treatment of <i>P. aeruginosa</i> infections.