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1. Abstract Biomedical Knowledge Graphs (BKGs) offer integrative representations of complex biology, yet their utility is compromised by the limitations of current construction methods: manual curation offers high fidelity but is unscalable, whereas purely automated Large Language Model (LLM) approaches often yield broad networks lacking mechanistic granularity. We present KG-Orchestra, an open-source multi-agent framework designed to build specialized, directional, cause-and-effect BKGs by enriching seed graphs. The framework focuses on increasing granularity within specific topics by leveraging Retrieval-Augmented Generation (RAG) to autonomously acquire, validate, and integrate evidence. The system orchestrates specialized agents for retrieval, schema alignment, and triplet validation with explicit, traceable provenance, transforming sparse seeds into dense, high-resolution resources. We evaluated KG-Orchestra on two specialized contexts—the mechanistic link between Nelivaptan and Alzheimer’s Disease (NADKG) and the complex probiotic interactions within the gut–brain axis (ProPreSyn-GBA)—across varying computational budgets. Our benchmarking results demonstrate that Qwen 3 variants deliver superior reasoning performance and that hybrid retrieval strategies significantly enhance evidence relevance. Furthermore, the multi-agent architecture ensures high triplet integrity and biological validity through iterative cross-checking and self-correction. The framework remains computationally flexible, deploying from single laptop GPUs to high-performance clusters. By bridging knowledge gaps and adding context-aware entities, KG-Orchestra increases reliability while validating seed assertions against up-to-date sources. This versatility supports critical downstream applications, including completing missing mechanistic pathways, integrating novel entities for drug repurposing, constructing targeted subgraphs from entity lists, and retroactively validating graph evidence for transparent auditing.