Drosophila melanogaster shares approximately 75% of human disease-related genes and possesses sophisticated genetic toolkits, including GAL4/UAS, CRISPR-Cas9, and RNA interference (RNAi), making it a rapid, cost-effective, and genetically tractable in vivo platform for natural products (NPs) discovery. This review systematically summarizes the modeling strategies, pathological mechanisms, and therapeutic applications of Drosophila models for six major human diseases, including type 2 diabetes, nephrolithiasis, inflammatory bowel disease, cancer, Alzheimer's disease, and Parkinson's disease. Cross-disease analysis identifies five evolutionarily conserved signaling networks-IIS/PI3K/Akt/FOXO, JNK/JAK/STAT, Nrf2/Keap1, mTOR/TORC1, and IMD/Toll-as common molecular targets of bioactive NPs, providing a unified mechanistic framework for understanding their multi-target pharmacological activities and broad therapeutic potential. Critically, we propose a generalizable integrated stepwise pipeline: high-throughput fly screening of crude extracts, bioassay-guided isolation of active monomers, genetic mechanistic dissection via RNAi and mutant rescue, and layered validation in human cells and selective mammalian models. This pipeline addresses key challenges in NPs research, including the identification of bioactive constituents and mechanistic validation, while improving screening efficiency and translational potential. Overall, this review establishes a multi-disease-applicable framework linking disease modeling, conserved signaling mechanisms, and translational pharmacology, providing practical guidance for future mechanism-driven NP discovery and preclinical development using Drosophila. By leveraging Drosophila genetics to bridge evolutionary conservation and human pathology, this framework offers a powerful, paradigm-shifting strategy to accelerate mechanism-driven NP discovery and preclinical development.
