Non-nutritive sweeteners (NNSs) are popular sugar substitutes, valued for their potential to reduce caloric intake and associated health risks. However, their long-term effects on the human gut microbiome remain debatable. This study investigates the impact of tagatose, allulose, Rebaudioside-A (Reb-A), and saccharin on quorum-sensing (QS)-regulated phenotypes and gene expression in QS biosensor model bacteria. It sheds light on their potential influence on the gut microbiome. Our study revealed diverse effects among the NNSs. Tagatose and allulose demonstrated QS phenotypic inhibition in Chromobacterium violaceum (≈50%) and Pseudomonas aeruginosa (20–50%) in a concentration-dependent manner. Additionally, tagatose and allulose decreased the P. aeruginosa lasI gene expression. Reb-A and saccharin presented a significant, however less prominent, phenotypic inhibition on C. violaceum (25–30%) and P. aeruginosa swarming motility (≈20%). Both NNSs decreased the expression of the lasI gene of P. aeruginosa. Molecular docking of QS regulatory proteins showed that saccharin and Reb-A have significantly higher binding affinity compared to allulose and tagatose, relative to native inducers. These results suggest the complex interactions mediated by NNSs in QS regulatory pathways. These findings provide valuable insights into the varied, species and dose-dependent effects of NNS on microbial communication, suggesting potential implications for the gut microbiome.