This study aimed to assess the efficacy of selenium nanoparticles (Se-NPs) in managing Fusarium wilt disease in lupine induced by Fusarium oxysporum f.sp. lupini. The study employed HR-TEM to characterize selenium nanoparticles, revealing a diverse morphology, uniform size distribution, and a mixture of crystalline and amorphous phases. The antifungal efficacy of Se-NPs (with an average particle size of 12-68 nm) at 5, 10, 15, 20, and 25 ppm was evaluated against the aggressive F. oxysporum isolate (Fox-Giza.1, accession No. PP230953.1). In vitro tests indicated that the Se-NPs at 25 ppm displayed the most significant inhibitory impact on mycelial growth, achieving a 100% decrease relative to the control. Anatomically, cross-sections of primary roots seen by scanning electron microscopy demonstrated that combining seed soaking and foliar spraying with Se-NPs treatment protected lupine plants against detrimental structural alterations caused by pathogens in the vascular cylinder structure. In vivo, testing performed in pots and fields demonstrated efficacy in combining seed soaking and foliar spray of Se-NPs treatment at 15 ppm for both, which was second only to the Topsin M-70® fungicide without significant differences, resulting in survived plants of 90.70%. In physiological studies, photosynthetic pigments, levels of peroxidase, polyphenol oxidase, chitinase, antioxidants capacity, total phenol, flavonoids, growth and yield metrics were increased in plants subjected to the successful combination treatment. In conclusion, the combination of seed soaking and foliar application of Se-NPs treatment were more effective than using each of them individually as a fungicide against Fusarium wilt disease in lupine.