To counteract bacterial resistance, we investigated the interruption of quorum sensing mediated by non-classical bioisosteres of the N-hexanoyl homoserine lactone with an azoline core. For this purpose, a set of selected 2-substituted azolines was synthesized, establishing the basis for a new protocol to synthesize 2-amino imidazolines. The synthesized compounds were evaluated as inhibitors of violacein production in Chromobacterium violaceum. Theoretical studies on bioisostere-protein interactions were performed using CviR. The results show that some azolines decreased violacein production, suggesting an antiquorum sensing profile against Gram-negative bacteria. Docking and molecular dynamic simulations together with binding free energy calculations revealed the exact binding and inhibitory profiles. These theoretical results show relationship with the in vitro activity of the azoline series.