Gray mold caused by Botrytis cinerea poses a major threat to tomato production worldwide. This study investigated the antifungal efficacy and defense-inducing potential of Agricultural Jiaosu (AJ), a fermented bioproduct derived from agricultural residues. In vitro, AJ exhibited strong inhibitory activity against B. cinerea (IC₅₀ = 3.9%), primarily through acidic metabolites (pH < 4.2) that disrupted fungal membranes and suppressed antioxidant enzymes, while later-stage inhibition was maintained by Acetobacter populations (6.7 × 10⁷ copies μL⁻¹) through competition for nutrients. In vivo, foliar application of 0.5% AJ significantly promoted tomato growth and enhanced resistance by stimulating antioxidant (SOD, CAT, POD) and defense-related (PAL, PPO) enzyme activities, reducing oxidative damage and lowering gray mold incidence by 55%. Collectively, AJ exerts a dual mode of action that combines direct pathogen suppression with activation of host systemic resistance. These results highlight AJ as a sustainable, residue-free biocontrol solution that offers an environmentally friendly alternative to chemical fungicides for effective management of gray mold in tomato cultivation.