Introduction Metabolite identification remains a major bottleneck in the understanding of metabolism. Many metabolomics studies end up with unknown compounds, leaving a landscape of metabolites and metabolic pathways to be unraveled. Therefore, identifying novel compounds within a metabolome is an entry point into the 'dark side' of metabolism. Objectives This work aimed at elucidating the structure of a novel metabolite that was first detected in the soil bacterium Acinetobacter baylyi ADP1 (ADP1). Methods We used high resolution multi-stage tandem mass spectrometry for characterizing the metabolite within the metabolome. We purified the molecule for 1D-and 2D-NMR (1 H, 13 C, 1 H-1 H-COSY, 1 H-13 C-HSQC, 1 H-13 C-HMBC and 1 H-15 N-HMBC) analyses. Synthetic standards were chemically prepared from MS and NMR data interpretation. Results We determined the de novo structure of a previously unreported metabolite: 3-((3-aminopropyl)amino)-4-hydroxy-benzoic acid. The proposed structure was validated by comparison to a synthetic standard. With a concentration in the mil-limolar range, this compound appears as a major metabolite in ADP1, which we anticipate to participate to an unsuspected metabolic pathway. This novel metabolite was also detected in another γ-proteobacterium. Conclusion Structure elucidation of this abundant and novel metabolite in ADP1 urges to decipher its biosynthetic pathway and cellular function.