<p>The microbiome of <i>Lilium pumilum</i> represents a valuable resource for developing sustainable biocontrol strategies. This study investigated the potential of these microorganisms to serve as major plant pathogen-antagonistic strains. In this study, 38 strains (18 fungi and 20 actinobacteria) were isolated from <i>L. pumilum</i> and its rhizosphere soil. Among these, the endophytic fungus Z-SD-LJ-2 (<i>Fusarium tricinctum</i>) exhibited remarkable broad-spectrum antifungal activity against five tested plant pathogens, with inhibition rates of 68.07–89.42% and half maximal effective concentration (EC₅₀) values of 16.58–30.97&#xa0;µg mL^− 1. Notably, its performance surpassed the commercial fungicide azoxystrobin against <i>Fusarium oxysporum</i> and <i>Botrytis cinerea</i>. Concurrently, the rhizosphere-derived strain Z-SDTR-2 (<i>Purpureocillium lilacinum</i>) demonstrated potent inhibition against <i>Rhizoctonia solani</i>, <i>Sclerotinia sclerotiorum</i>, and <i>Botrytis cinerea</i>, with all inhibition rates exceeding 80% and EC₅₀ values of 17.17–21.80&#xa0;µg mL^− 1. Activity-guided isolation from Z-SD-LJ-2 led to the identification of enniatin B as the most active compound, exhibiting EC₅₀ values of 13.78–26.81&#xa0;µg mL^− 1. Further studies revealed that enniatin B induces apoptosis in the pathogens by triggering reactive oxygen species (ROS) accumulation and causing mitochondrial dysfunction. In pot experiments, the fermentation broth of Z-SD-LJ-2 (2 × 10³ mg L^− 1) effectively controlled lily wilt, reducing the disease index to 30% (14.3% lower than azoxystrobin) and achieving a preventive efficacy of 72.6% (18% higher than the control). Additionally, the treatment promoted lily growth, increasing plant height by 4.4% and stem thickness by 8.71% compared to the control, underscoring its dual-function potential as a novel, effective biopesticide for sustainable agricultural practices.</p>