In the present study, Trichoderma atroviride TRS25 is presented as a biological control agent, which significantly limits the development of infection and reduces the disease caused by the pathogenic fungus Rhizoctonia solani in cucumber plants ( Cucumis sativus L.). The systemic disease suppression is related to oxidative, signaling, and biochemical changes, that are triggered in response to a pathogen. Induction of systemic defense in cucumber by TRS25 greatly depends on the accumulation of signaling molecules including hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) as well as salicylic acid (SA) and its derivatives including methyl salicylate (MeSA) and octyl salicylate (OSA). The study established that NO was accumulated in leaves and shoots of the cucumber plants, especially those pretreated with Trichoderma and inoculated with R. solani , where the compound was accumulated mainly in the cells localized in the vascular bundles and in epidermal tissues. We suggest, for the first time, that in the plants pretreated with TRS25, the accumulation of H 2 O 2 and NO may be related to catalase (CAT) and S-nitrosoglutathione reductase (GSNOR) activity decrease. On the other hand, excessive accumulation of NO and SA may be controlled by forming their inactive forms, S-nitrosothiols (SNO) and salicylic acid glucosylated conjugates (SAGC), respectively. The obtained results suggest that the mentioned molecules may be an important component of the complex signaling network activated by TRS25, which is positively involved in systemic defense responses of cucumber plants against R. solani .