Soil salinity poses a significant challenge in agriculture, disrupting the normal functioning of plants by reducing water and nutrient uptake. Olive trees (Olea europaea), common in Mediterranean regions, exhibit moderate to high tolerance to salinity, varying by cultivar. Interest in cultivating olive trees is growing in Florida’s coastal areas, characterized by poorly drained soil and low-quality groundwater, leading to salt accumulation in the root zone. The high salinity levels in these areas present a significant challenge for crop cultivation. Therefore, introducing new salt-tolerant cultivars is necessary to mitigate salinity stress. This study aimed to evaluate the plant physiological and root anatomical responses of two novel olive cultivars - ‘Oliana’ and ‘Lecciana’ - to salinity stress, assessing their salt tolerance. Eight-month-old plants were grown in pots using a sand medium under greenhouse conditions and treated with varying salt concentrations (0 mM, 50 mM, and 100 mM). The experiment followed a completely randomized design with three replications, each consisting of nine plants. Plants were irrigated at weekly intervals with half-strength Hoagland solution to meet their nutrient requirements. Height and trunk diameter were measured at four different time points (0, 15, 30, and 45 days). At the end of the trial, plants were destructively sampled for biomass, nutrient content, and root anatomical measurements at the latter three time points. Significant differences were observed in height, trunk diameter, and nutrient contents between the control and NaCl treatments. These findings serve as a baseline for the commercial development of salt-tolerant olive cultivars.