Biofilm formation plays a key role in many bacteria causing infections, which mostly accounts for high-frequency infectious recurrence and antibiotics resistance. In this study, we sought to compare modified metabolism of biofilm and planktonic populations with UTI89, a predominant agent of urinary tract infection, by combining mass spectrometry based untargeted and targeted metabolomics methods, as well as cytological visualization, which enable us to identify the driven metabolites and associated metabolic pathways underlying biofilm formation. Surprisingly, our finding revealed distinct differences in both phenotypic morphology and metabolism between two patterns. Furthermore, we identified and characterized 38 differential metabolites and associated three metabolic pathways involving glycerolipid metabolism, amino acid metabolism and carbohydrate metabolism that were altered mostly during biofilm formation. This discovery in metabolic phenotyping permitted biofilm formation shall provide us a novel insight into the dissociation of biofilm, which enable to develop novel biofilm based treatments against pathogen causing infections, with lower antibiotic resistance.