Selenium is an essential micronutrient that provides important benefits to plants and humans. At proper concentrations, selenium increases plant growth, pollen vitality, the shelf life of fresh products, and seems to improve stress resistance; these effects can certainly be attributed to its direct and indirect antioxidant capacity. For these reasons, in the present work, the effects of selenium at different dosages on in vitro cultivated olive explants were investigated to observe possible positive effects (in terms of growth and vigor) on the proliferation phase. The work was carried out on four different olive cultivars: “San Felice”, “Canino”, “Frantoio”, and “Moraiolo”. The explants were cultured in aseptic conditions on olive medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. The experimental scheme included a comparison between explants grown with five different concentrations of Na2SeO4 (0, 10, 20, 40, and 80 mg L−1) added to the medium during three successive subcultures. Interesting information has emerged from the results and all varieties responded to different concentrations of Selenium. The optimal Se dosages varied for each cultivar, but in general, Se concentration between 10 and 40 mg L−1 increased fresh and dry weight of the explants and shoot lengths. Se treatment induced in all cultivars and for all dosages used an increase in total Se content in proliferated explants. Furthermore, as the subcultures proceeded, the ability of the explants to absorb Se did not diminish. The Se content ranged from 8.55 to 114.21 µg kg−1 plant DW in ‘Frantoio’, from 9.83 to 94.85 µg kg−1 plant DW in ‘Moraiolo’, from 19.84 to 114.21 µg kg−1 plant DW in ‘Canino’, and from 20.97 to 95.54 µg kg−1 plant DW in ‘San Felice’. In general, the effect of selenium tends to decrease with the progress of subcultures and this suggests a sort of “adaptation” effect of the explants to its presence. The present study highlights for the first time the possibility of using in vitro cultures as biotechnological support to study supplementation with selenium and its effects on in vitro olive plant growth.