Powdery mildew caused by Sphaerotheca fusca poses a significant threat to cucumber (Cucumis sativus L.) production worldwide, underscoring the need for sustainable disease management strategies. This study investigates the potential of L-lysine, abundantly produced by Bacillus subtilis M 320 (BSM320), to prime systemic acquired resistance (SAR) pathways in cucumber plants. Liquid chromatography–mass spectrometry analysis identified L-lysine as the primary bioactive metabolite in the BSM320 culture filtrate. Foliar application of purified L-lysine significantly reduced powdery mildew symptoms, lowering disease severity by up to 92% at concentrations ≥ 2500 mg/L. However, in vitro spore germination assays indicated that L-lysine did not exhibit direct antifungal activity, indicating that its protective effect is likely mediated through the activation of plant immune responses. Quantitative reverse transcription PCR revealed marked upregulation of key defense-related genes encoding pathogenesis-related proteins 1 and 3, lipoxygenase 1 and 23, WRKY transcription factor 20, and L-type lectin receptor kinase 6.1 within 24 h of treatment. Concurrently, salicylic acid (SA) levels increased threefold in lysine-treated plants, confirming the induction of an SA-dependent SAR pathway. These findings highlight L-lysine as a sustainable, residue-free priming agent capable of enhancing broad-spectrum plant immunity, offering a promising approach for amino acid-based crop protection.