Bacteria use small signaling molecules to communicate in a process termed “quorum sensing” (QS), which enables the coordination of survival strategies, such as production of virulence factors and biofilm formation. In Gram‐negative bacteria, these signaling molecules are a series of N‐acylated L‐homoserine lactones. With the goal of identifying non‐native compounds capable of modulating bacterial QS, a virtual library of N‐dipeptido L‐homoserine lactones was screened in silico with two different crystal structures of LasR. The 30 most promising hits were synthesized on HMBA‐functionalized PEGA resin and released through an efficient acid‐mediated cyclative release mechanism. Subsequent screening for modulation of QS in Pseudomonas aeruginosa and E. coli identified six moderately strong activators. A follow‐up library designed from the preliminary derived structure–activity relationships was synthesized and evaluated for their ability to activate the QS system in this bacterium. This resulted in the identification of another six QS activators (two with low micromolar activity) thus illuminating structural features required for QS modulation.