Here we report the molecular basis of tolaasin biosynthesis in Pseudomonas costantinii and show by imaging mass spectrometry that the toxic lipopeptides are produced during infection of the button mushroom Agaricus bisporus. To unravel the molecular basis of toxin biosynthesis, we subjected genomic DNA of P. costantinii to shotgun sequencing, identified a large nonribosomal peptide synthetase (NRPS) gene cluster (comprising 18 biosynthetic modules), and confirmed its function by targeted gene knockout (ΔtaaB mutant, which abolished tolaasin production and virulence). Bioinformatic analysis of A domain specificities correlated with the primary sequence of tolaasins. MS/MS fragmentation, high-resolution MS, and isotope-labeling studies (e.g., [1-¹³C]L-leucine, L-Val 2d¹) determined the main tolaasin derivative from P. costantinii as tolaasin F (differing from tolaasin I at amino acid position 15 (Leu vs. Ile) and Val9 configuration (D- instead of L-)). Phylogenetic analysis of condensation (C) domains revealed a novel subgroup of pseudo-D CL domains (exhibiting a QQ(Q/H) motif and lacking epimerization activity), refining Balibar's rule. MALDI imaging mass spectrometry confirmed that tolaasins are produced in situ during infection of A. bisporus. In conclusion, we elucidated the entire biosynthetic gene cluster coding for tolaasin in P. costantinii and other pseudomonads, verified its role in virulence, and improved the prediction of amino acid configuration in nonribosomally synthesized peptides. This study provides insights into the pathobiology of toxinogenic pseudomonads and a foundation for the development of biocontrol methods against brown blotch disease.