To tackle selenium (Se) malnutrition, biofortification is among the proposed strategies. A biostimulant application in soils is thought to support a plant’s growth and productivity. Biofortification with Se(VI) may lead to a leaching hazard due to the high mobility of Se(VI) in the soil environment. In this study, the effect of the application of two Se(VI) rates—5 and 10 mg kg−1 soil—and a biostimulant on the Se uptake by lettuce plants and on the Se(VI) distribution in soil fractions following the plants’ harvest, was investigated. Phosphorus (P) and sulfur (S) concentrations in plants were also determined. A high Se(VI) rate suppressed plant growth, leading to a significant fresh weight decrease from 12.28 to 7.55 g and from 14.6 to 2.43 g for the control and high Se(VI) without and with biostimulants, respectively. Impaired plant growth was verified by the SPAD, NDVI and NDRE measurements. The significantly highest Se concentration in plants, 325 mg kg−1, was recorded for the high Se(VI) rate in the presence of the biostimulant. Compared to controls, the low Se(VI) rate significantly decreased P and increased the S concentrations in plants. The post-harvest soil fractionation revealed that, in the presence of the biostimulant, the Se(VI) soluble fraction increased from 0.992 to 1.3 mg kg−1 at a low Se(VI) rate, and decreased from 3.T85 to 3.13 mg kg−1 at a high Se(VI) rate. Nevertheless, at a low Se(VI) rate, 3.6 and 3.1 mg kg−1 of the added Se(VI) remained in the soil in less mobile forms, in the presence or absence of the biostimulant, respectively. This study indicated that the exogenous application of Se in soil exerted dual effects on lettuce growth and Se availability, depending on the level of selenate applied.