Background <p>Dendrobine, a neuroprotective and anticancer sesquiterpenic alkaloid, is primarily sourced from endangered <i>Dendrobium</i> orchids, posing sustainability challenges to its production. Endophytic fungi, such as <i>Trichoderma longibrachiatum</i> MD33, offer an alternative; however, unresolved biosynthetic pathways and low yields hinder industrial scalability. Enhancing fungal metabolism through nanotechnology could address these limitations; however, nanoparticle-mediated engineering remains unexplored for dendrobine biosynthesis. This study aimed to (1) optimize dendrobine production in <i>T. longibrachiatum</i> MD33 using gold nanoparticles (CH-AuNPs) functionalized with alkaloid precursors and (2) elucidate the biosynthetic pathway to enable targeted metabolic engineering. CH-AuNPs were chemically synthesized, functionalized with L-phenylalanine, L-tyrosine, and tyramine, and applied to fungal cultures at concentrations of 0.5–20.0 mg/L. Multi-omics analyses (transcriptomics, proteomics, and metabolomics) identified pathway enzymes, and oxidative stress markers and dendrobine yields were quantified.</p> Results <p>Dose-dependent CH-AuNP exposure (10.0&#xa0;mg/L optimal) elevated dendrobine production by 63.7%, balancing pathway activation and oxidative stress. Multi-omics analysis revealed a hybrid terpenoid-alkaloid pathway, wherein sesquiterpene scaffolds from the mevalonate pathway merge with ornithine-derived piperidine moieties. This process is regulated by sesquiterpene synthases (<i>TPS</i>), cytochrome P450s (<i>CYP71D1</i>), and O-methyltransferases (<i>COMT</i>). Metabolomic analysis provided direct evidence for the rechanneling of nitrogen metabolism, with depletion of glutamate and ornithine pools and accumulation of polyamine pathway intermediates such as putrescine, supporting the transcriptional upregulation of ornithine decarboxylase (ODC). Mechanistically, low-to-moderate oxidative stress induced by CH-AuNPs activated redox-sensitive transcription factors and stress-responsive pathways, which in turn upregulated terpenoid and alkaloid biosynthesis genes. This controlled stress response enhanced precursor flux and enzyme activity, leading to increased dendrobine synthesis without triggering cellular damage in the cells. Concentrations &gt; 10.0&#xa0;mg/L suppressed metabolism owing to oxidative damage.</p> Conclusions <p>CH-AuNPs act as precision tools to upregulate dendrobine biosynthesis in <i>T. longibrachiatum</i> MD33, resolving the hybrid pathway and establishing this fungus as a sustainable production platform for dendrobine. The dose-dependent response highlights the dual role of nanoparticle-mediated engineering in metabolic enhancement and stress induction. This integration of nanotechnology and multi-omics bridges the critical gaps in fungal biotechnology, enabling scalable and eco-friendly alkaloid synthesis. Future applications include CRISPR-AuNP genome editing and bioreactor optimization, which will advance pharmaceutical and environmental biotechnologies.</p>