Soil salinity is a major constraint on global agricultural productivity. This study evaluated the efficacy of a cell-free extract from Trichoderma hamatum (designated BEYF) in enhancing salt stress tolerance in lettuce (Lactuca sativa). Lettuce plants under normal and salt-stressed conditions exposed to 200 mM NaCl were treated with either water or YF (the working solution of BEYF) at concentrations of 0.05, 0.10, and 0.25 mg/L. Compared to the control, YF application significantly improved plant growth under salt stress, as indicated by increased plant height, biomass, leaf area, and other agronomic traits. Physiologically, YF mitigated oxidative membrane damage, as indicated by reduced electrolyte leakage and malondialdehyde (MDA) content, while promoting the accumulation of the osmoprotectant proline. Histochemical staining further confirmed that YF effectively suppressed hydrogen peroxide (H₂O₂) accumulation and preserved cell viability under salt stress. At the molecular level, YF significantly up-regulated the expression of key stress-responsive genes, including those involved in abscisic acid biosynthesis (NCED1, NCED2), signaling (WRKY58), and proline synthesis (P5CSs). Collectively, our findings demonstrate that BEYF enhances lettuce salt tolerance through integrated physiological, cellular, and transcriptional adaptations, supporting its potential as a sustainable biostimulant for improving crop cultivation in saline soils.