Background <p>Plant-derived peptides can act as resistance inducers and represent promising tools for sustainable crop protection. Despite growing interest and application, their broader effects on plant-associated microbiomes remain insufficiently characterized. Here, we investigated the impact of an immunomodulatory peptide derived from the tomato defense protein Prosystemin on the tomato phyllosphere microbiome and leaf volatilome.</p> Results <p>The peptide was applied as a foliar spray at biweekly intervals from planting to two months post-germination to approximate common agricultural practices. Shotgun metagenomic sequencing combined with qPCR revealed abundant bacterial communities (up to 4.6 log₁₀ bacterial 16S rRNA gene copies) dominated by Actino-, Alphaproteo- and Gammaproteobacteria across all samples. Peptide treatment was associated with a significant shift in community structure, characterized by reduced alpha diversity and increased microbial associations. Several genera, including <i>Acinetobacter</i>, <i>Sphingobium</i>, <i>Sphingomonas</i>, <i>Brevundimonas</i>, and <i>Massilia</i>, increased in relative abundance following treatment. Functional profiling indicated rearrangements in gene categories related to stress response and metabolic adaptation. Notably, volatilome analysis further revealed elevated monoterpene emissions in peptide treated plants, consistent with activation of defense-associated metabolism. Members of the Sphingomonadaceae family, particularly <i>Sphingobium yanoikuyae</i>, appear well suited to persist under peptide-associated conditions and may therefore contribute to the observed community restructuring, although causal mechanisms remain to be tested.</p> Conclusion <p>Beyond its established role in protecting tomato against pests and necrotrophic fungi, the Prosystemin-derived peptide provides an opportunity to investigate peptide-triggered plant responses and their interactions with the plant microbiota.</p>