In this study, we synthesized four series of novel L-homoserine lactone analogs and evaluated their in vitro quorum sensing (QS) inhibitory activity against two biomonitor strains, Chromobacterium violaceum CV026 and Pseudomonas aeruginosa PAO1. Studies of the structure–activity relationships of the set of L-homoserine lactone analogs indicated that phenylurea-containing N -dithiocarbamated homoserine lactones are more potent than ( Z )-4-bromo-5-(bromomethylene)-2(5 H )-furanone ( C30 ), a positive control for biofilm formation. In particular, compared with C30 , QS inhibitor 11f significantly reduced the production of virulence factors (pyocyanin, elastase and rhamnolipid), swarming motility, the formation of biofilm and the mRNA level of QS-related genes regulated by the QS system of PAO1. These results reveal 11f as a potential lead compound for developing novel antibacterial quorum sensing inhibitors.