Citrus fruits are among the largest fruit crops in the world. These crops are exposed to several challenges that cause severe yield losses due to abiotic stress each year. Where water stress is a major environmental factor, breeding approaches need to develop tolerant rootstocks that can mitigate risks from climate and other key abiotic factors. In this work, the impact of two Trehalase enzyme inhibitors, Validamycin and Zn²⁺, on increasing Trehalose accumulation and inducing salinity tolerance in two citrus rootstocks, sour orange and Troyer citrange, was studied. Our findings suggest that application of pre-soaking of seeds with 30 μM of Validamycin A and 20 mM ZnSO4 alleviated the adverse effects of salinity by stimulating growth, increasing total chlorophyll content, and reducing antioxidant enzyme activity under severe salt stress conditions (50 mMNaCl) compared with the untreated control. An additional point of interest was to detect the change in the expression of the Trehalose gene in response to salt stress with or without Val/Zn treatment.