The effects of a fungal elicitor from Trichothecium roseum on signal pathways of salicylic acid (SA), jasmonic acid (JA), and Ca²⁺ in potato tubers were investigated. The results showed that fungal elicitor treatment effectively inhibited the lesion diameter of Fusarium sulphureum in vivo, which was 17.5% lower than that of the control. In addition, fungal elicitor treatment triggered an increase in O₂⁻ production and H₂O₂ content. The fungal elicitor enhanced the activities and gene expression levels of isochorismate synthase (ICS), phenylalanine ammonia lyase (PAL), allene oxide cyclase (AOC), allene oxide synthase (AOS), lipoxygenase (LOX), and Ca²⁺-ATPase. Furthermore, the fungal elicitor promoted an increase in calmodulin (CaM) content. Protective enzymes (dismutase (SOD), catalase (CAT), polyphenol oxidase (PPO), chitinase (CHI), and β-1,3-glucanase (Glu)) and disease-resistance-related genes (PR1, PR2, and PDF1.2) were induced to be upregulated by elicitor treatment. These results indicated that the fungal elicitor induced disease resistance by accelerating the accumulation of reactive oxygen species (ROS), activating SA, JA, and Ca²⁺ signaling, and upregulating resistance genes. The results of this study revealed the molecular mechanism of fungal elicitor-induced resistance in the potato, which provides a theoretical basis for the mining of new, safe, and efficient elicitor-sourced antifungal agents and is of great importance for the effective control of potato dry rot disease.