The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone. Newly synthesized compounds were a series of analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, and the structures of all six synthesized compounds was confirmed by NMR and GC/MS analyses. These new QS inhibitor candidates could remarkably inhibit both Pseudomonas QS signaling and biofilm formation, which were assayed by using the recombinant reporter system and flow cell confocal microscopy. The degree of QS inhibition by these new inhibitors varied from 20% to 90%. For the profound understanding about inhibition mechanism, we tried to estimate the binding energy between QS receptor, LasR, and our inhibitors from the in silico modeling system. The predicted binding pattern from the modeling system and our experimental data about QS inhibition were in good agreement. From these results, we suggest a new approach to develop the QS inhibitors and biofilm control agents based on structural modeling.