Wheat (Triticum aestivum) employs various strategies to defend against Fusarium oxysporum, a soil-borne vascular fungal pathogen that disrupts structural integrity and metabolism. The purpose of this research was to ascertain the alterations of anatomical and biochemical responses in wild-type (WT) and DPA-treated wheat (T. aestivum) seedlings exposed to F. oxysporum. The WT and DPA-treated seedlings showed disorganization of parenchyma cells, sclerenchyma cells, vascular bundles (VBs), and lower numbers of xylem (Xy) and phloem (Ph) cells, and reduced thickness of the cuticle layer (C) at the epidermal layer of shoots. The content of chlorophyll (Chl), carbohydrate, and nucleic acid was reduced in WT and DPA-treated seedlings during infection. Enhanced defense responses through peroxidase (POD), and polyphenol oxidase (PPO) was observed to be high in WT as compared to DPA-treated seedlings under stress condition. In addition, the content of salicylic acid (SA) and phenolics was increased in WT than DPA under stress condition. However, the DPA-treated seedlings showed enhanced growth of fungal mycelia compared to WT during stress condition. Hence, the anatomical and biochemical aspects of DPA-treated seedlings decreased as compared to WT when exposed to F. oxysporum.