Phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) is a highly conserved, multistep chemical process which uses phosphate transfer to regulate the intake and use of sugars and other carbohydrates by bacteria. In addition to controlling sugar uptake, the PTS regulates several bacterial cellular functions such as chemotaxis, glycogen metabolism, catabolite repression and biofilm formation. Previous studies have shown that the phosphoenolpyruvate (PEP) to pyruvate ratio is a critical determinant of PTS functions. This study shows that 2-oxo-4-phenyl-2,5-dihydro-3-furancarbonitrile (MW01), a compound with structural similarity to known natural products, induces Vibrio cholerae to grow preferentially in the biofilm mode in a mechanism that involves interaction with pyruvate. Spectrophotometric assays were used to monitor bacterial growth kinetics in microtiter plates and quantitatively evaluate biofilm formation in borosilicate glass tubes. Evidence of MW01 and pyruvate interactions was determined by nuclear magnetic resonance spectroscopy. Given the established connection between PTS activity and biofilm formation, this study also highlights the potential impact that small-molecule modulators of the PTS may have in the development of innovative approaches to manage desired and undesired microbial cultures in clinical, industrial and environmental settings.