Acremonium wilt disease affects grain quality and reduces sorghum yield around the globe. The present study aimed to assess the efficacy of humic acid (HA)-coated Fe3O4 (Fe3O4/HA) nanoparticles (NPs) in controlling acremonium wilt disease and improving sorghum growth and yields. During the season 2019, twenty-one sorghum genotypes were screened to assess their response to Acremonium striticum via artificial infection under field conditions and each genotype was assigned to one of six groups, ranging from highly susceptible to highly resistant. Subsequently, over the two successive seasons 2020 and 2021, three different concentrations of 10, 40 and 80 mg L−1 of Fe3O4/HA NPs were tested against A. striticum. The concentrations of 40 and 80 mg L−1 were found to be highly effective in controlling acremonium wilt disease on different sorghum genotypes: LG1 (highly susceptible), Giza-3 (susceptible), and Local 119 (resistant) genotypes. After harvest, the physiological (growth and yield) and biochemical (peroxidase, catalase, and gibberellic acid) attributes of sorghum plants were determined, and the results demonstrated that concentrations of 40 and 80 mg L−1 increased peroxidase and catalase activities in healthy (uninoculated) sorghum genotypes compared to inoculated sorghum genotypes. Additionally, the toxicity of Fe3O4/HA NPs on male albino rats was investigated via hematological (CBC), chemical (ALT and AST) and histopathological analyses. The concentration 80 mg L−1 of Fe3O4/HA NPs caused a marked increase in ALT and creatinine level after 51 days of feeding. Severe pathological alterations were also observed in liver and kidney tissues of rats administered with grain sorghums treated with 80 mg L−1. In comparison with the untreated control plants, a concentration of 40 mg L−1 significantly increased the growth, yield and gibberellic acid levels (p ≤ 0.05) and was found to be safe in male albino rats. Conclusively, a concentration of 40 mg L−1 of Fe3O4/HA NPs showed promising results in curtailing A. striticum infections in sorghum, indicating its great potential to substitute harmful fertilizers and fungicides as a smart agriculture strategy.