Abstract The vascular wilting caused by Fusarium oxysporum f. sp. dianthi (Fod) is the most relevant disease for carnation cultivation. Understanding the biochemical mechanisms involved in resistance to Fod will allow the development of new disease control strategies. In this research, the levels of some phytohormones such as salicylic acid (SA), methyl salicylate (MeSA), and methyl jasmonate (MeJA) were evaluated in symplast of carnation roots infected with this pathogen. The accumulation of these hormones was then correlated with the expression levels of symplastic peroxidases, enzymes involved in the plant resistance against pathogen during interaction. Our results suggested that pathogen infection causes a differential accumulation of SA, MeSA, and MeJA in a resistant cultivar (i.e. ‘Golem’), being earlier and higher than that observed in a susceptible one (i.e. ‘Solex’). Simultaneously, an increase of guaiacol peroxidase enzymatic activity (GPX) and transcriptional levels of a gene coding for a symplastic peroxidase were presented as part of the resistance response. The positive statistical correlation between the accumulation of SA and MeJA and the expression of peroxidases (GPX activity and mRNA levels) indicates the possible cellular relationship of these phenomena during the activation of the resistance to Fod. Our findings suggested some hormonal signaling mechanisms acting at the roots during the regulation of the biochemical response associated with resistance against Fod.