Pseudostellaria heterophylla (Miq.) Pax ex Pax et Hoffm. is a member of the Caryophyllaceae family, in which dried tuberous root is the well-known traditional Chinese medicine (TCM) and a widespread food ingredient in Asia. In recent years, the large-scale cultivation of P. heterophylla has led to frequent infectious diseases caused by multiple pathogens. However, efficient and safe approaches for preventing and managing P. heterophylla diseases have become urgent for this high-quality industrial development. Herein, a culturable microbiome of diseased P. heterophylla rhizosphere soil was constructed, and the broad-spectrum antifungal activity of Pseudomonas was screened. Three P. palleroniana strains, B-BH16-1, B-JK4-1, and HP-YBB-1B, were isolated and identified with vigorous antifungal activity by confrontation method. We employed the PacBio RS II single-molecule real-time (SMRT) sequencing and Illumina sequencing methods to obtain the genome of these three isolates. Phylogenetic, synteny, and ANI analysis showed that the lineage between strain B-JK4-1 with B-BH16-1 or HY-YBB-1B was closer than that between strain B-BH16-1 with HP-YBB-1B. The comparative genome of strains B-BH16-1, B-JK4-1, and HP-YBB-1B showed marked differences in secondary metabolite biosynthesis genes among these three P. palleroniana strains. Strain B-BH16-1, B-JK4-1, and HP-YBB-1 produced tolaasin I/tolaasin F (23 genes), sessilin A (37 genes), and putisolvin (39 genes), respectively. CAZyme analysis showed that 126, 129, and 127 CAZymes were identified in strains B-BH16-1, B-JK4-1, and HP-YBB-1B genomes, which genes in auxiliary activities (AA), carbohydrate esterases (CE), and glycosyl transferases (GT) categories were different among these three strains. These results provide new insights into the divergent antifungal metabolites in pathogen-driven three P. palleroniana strains against primary pathogens of Pseudostellaria heterophylla.