Tobacco root rot (TRR), caused by Fusarium oxysporum , poses a significant threat to tobacco production. Synthetic fungicides widely used to control this soil-borne pathogen frequently cause fungicide resistance and environmental contamination. Limonene, a natural terpene with potent antifungal activity, features low residue and high biosafety, rendering it a promising natural substitute for chemical fungicides against TRR. This study aimed to investigate the antifungal activity and mechanisms of limonene against F. oxysporum using in vitro assays, physiological tests, transcriptomics, and metabolomics. The results demonstrated that limonene significantly inhibited mycelial growth and spore proliferation, with an EC 50 of 201.2 μg/mL (1.48 mM). It exhibited a dose-dependent repellent effect on the chemotactic behavior of F. oxysporum , with effective concentrations as low as 25 μg/mL (0.18 mM). Furthermore, it disrupted cell membrane integrity and permeability at concentrations as low as 100 μg/mL (0.73 mM). Transcriptomic screening identified 3,871 differentially expressed genes (DEGs), which were significantly enriched in ribosome, proteasome, ER protein processing, autophagy and glyoxylate metabolism pathways. Eight representative key DEGs ( cat1 , rhogap , tf , cyc , bro1 , mic12 , rpl13 , clptm1l ) related to fungal growth and stress response were verified by RT-qPCR verification, and their expression trends were consistent with the transcriptomic results. Metabolomic analysis revealed 626 differential accumulated metabolites (DAMs), three key metabolites (ortho-Hydroxyphenylacetic acid, 4-Methylphenol and Lenticin) were significantly altered under limonene treatment. These DAMs were mainly enriched in starch and sucrose, riboflavin, folate and tyrosine metabolism pathways. Multi-omics joint analysis indicated that key targeted pathways including energy metabolism, amino acid metabolism, lipid metabolism, and protein synthesis and degradation were regulated. In conclusion, limonene inhibits F. oxysporum through multiple mechanisms, including cell membrane damage, chemotaxis blockage, transcriptional regulation, and metabolic disturbance. This study provides scientific support for the development of limonene as a green botanical fungicide for controlling tobacco root rot.