Iodine (I) and selenium (Se) are included in the group of beneficial elements. They both play important roles in humans and other animals, particularly in the regulation of thyroid functioning. A substantial percentage of people around the world suffer from health disorders related to the deficiency of these elements in the diet. Salicylic acid (SA) is a compound similar to phytohormones and is known to improve the efficiency of I biofortification of plants. The influence of SA on Se enrichment of plants has not, however, been recognized together with its effect on simultaneous application of I and Se to plants. Two-year studies (2014-2015) were conducted in a greenhouse with hydroponic cultivation of lettuce in an NFT (nutrient film technique) system. They included the application of I (as KIO3), Se (as Na2SeO3) and SA into the nutrient solution. KIO3 was used at a dose of 5 mg I⋅dm(-3) (i.e., 39.4 μM I), while Na2SeO3 was 0.5 mg Se⋅dm(-3) (i.e., 6.3 μM Se). SA was introduced at three doses: 0.1, 1.0, and 10.0 mg⋅dm(-3) nutrient solutions, equivalent to 0.724, 7.24, and 72.4 μM SA, respectively. The tested combinations were as follows: (1) control, (2) I + Se, (3) I + Se + 0.1 mg SA⋅dm(-3), (4) I + Se + 1.0 mg SA⋅dm(-3) and (5) I + Se + 10.0 mg SA⋅dm(-3). The applied treatments had no significant impact on lettuce biomass (leaves and roots). Depending on the dose, a diverse influence of SA was noted with respect to the efficiency of I and Se biofortification; chemical composition of leaves; and mineral nutrition of lettuce plants, including the content of macro- and microelements and selenocysteine methyltransferase (SMT) gene expression. SA application at all tested doses comparably increased the level of selenomethionine (SeMet) and decreased the content of SA in leaves.