Quorum sensing (QS) plays an essential role in biofilm formation, a critical, multi-step, and complex process in the development of dental plaque. QS regulates the expression of virulence factors, aggregation, and bacterial adhesion within the biofilm. Scientifically known as 4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose or 1,6-dichloro-1, 6-dideoxy-β-d-fructofuranosyl 4-chloro-4-deoxy-α-d-galacto-pyranoside, sucralose was synthesized by substituting three hydroxyl groups in sucrose at positions 4, 1', and 6' with chlorines. When compared to sucrose, sucralose is hundreds of times sweeter (approximately 600 times). Unlike sucrose, sucralose is a non-cariogenic artificial sweetener, commonly included in dental care products such as chewing gums, toothpastes, and mouthrinses to enhance palatability for consumers. While its non-cariogenic action is well established, there is limited evidence regarding the potential anti-cariogenic mechanisms of sucralose. This study investigated whether sucralose interferes with QS involved in oral bacterial biofilm formation. A representative LuxR-type quorum sensing regulator, LasR, was expressed in the presence of sucralose and/or its native ligand, N-acyl homoserine lactone (AHL). The expressed protein was purified using nickel-affinity chromatography and quantified by the Bradford assay. The findings reveal that sucralose significantly inhibits AHL-dependent signaling, presumably by disrupting receptor–ligand interactions. These results provide insights into a possible molecular mechanism underlying the anti-cariogenic action of sucralose, highlighting its potential as a functional additive in oral health formulations.