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Background Plant-herbivorous insect interactions are crucial for deciphering plant defence strategies and maintaining ecosystem functions. However, how tree species richness influences the anti-herbivore defence of extrafloral nectary-bearing trees in subtropical forests remains elusive. We conducted a field experiment to assess the responses of seven biosynthetic “pathway”-level metabolites in extrafloral nectary-bearing trees and phyllosphere microbiome to herbivorous insect-induced leaf damage across subtropical forest communities with varying tree species richness (i.e. monoculture, two-, and four-tree species mixtures).Results Our results revealed that the effect of leaf damage severity on metabolite diversity was dependent on tree species richness. Specifically, leaf damage severity positively affected the abundance of defensive metabolites in monoculture plots, whereas leaves with greater damage severity exhibited higher diversity of defensive metabolites in mixture plots. With increasing leaf damage severity, bacterial diversity and the number of keystone taxa in co-occurrence networks increased, while network complexity decreased. Notably, compared to fungal communities, bacterial diversity, bacterial keystone taxa, and bacterial interactions potentially mediated changes in defensive metabolites.Conclusions Our findings highlight plant diversity-dependent synergies between herbivory pressure and phyllosphere bacterial communities in shaping plant chemical defences, providing insights into plant-microbe-herbivorous insect relationships.