Luffa (Luffa aegyptiaca) is an economically important crop cultivated for its vegetable, fibrous, and bioactive properties, yet its productivity is severely constrained by Fusarium diseases. This study aimed to evaluate the resistance responses of eight luffa genotypes—comprising five indigenous Iranian accessions (Northern Black Seed, Angled, Northern White Seed, Touri, and Northern Large) and three non-indigenous accessions (Afghani, Long luffa, and Brazilian)—against a Fusarium isolate obtained from melon fields in Iran. Pathogenicity assays were conducted using the root-dip inoculation method with a conidial suspension (1 × 10⁶ spores mL⁻¹), and disease progression was monitored for two months. Disease severity was quantified using disease index (DI) and area under the disease progress curve (AUDPC). Significant genotypic differences in disease response were observed. Three genotypes—Brazilian, Northern Black Seed, and Northern White Seed—exhibited complete resistance (DI = 0%; AUDPC = 0), showing no visible symptoms throughout the evaluation period. Three additional genotypes (Angled, Long luffa, and Afghani) demonstrated partial resistance with low DI values (0–15%), while Northern Large and Touri were classified as sensitive, exhibiting DI values of 64% and 44%, respectively. No genotype displayed highly sensitive responses. These findings identify valuable sources of genetic resistance, particularly the completely resistant indigenous genotypes, which represent promising candidates for integration into breeding programs aimed at developing Fusarium-resistant luffa cultivars. The resistant accessions may harbour effective defense mechanisms, including physical and chemical barriers, that restrict pathogen establishment and progression. This study provides foundational knowledge for sustainable luffa production through genetic improvement and emphasizes the necessity of conserving and utilizing indigenous germplasm as reservoirs of disease resistance traits.