The aim of the study was to determine the effect of biostimulants on the content and uptake of selected macronutrients (phosphorus, calcium, magnesium and potassium) in tubers of two varieties of Jerusalem artichoke. The experiment was conducted in a two-factor arrangement (split-plot) with three replications in 2021-2022 at an individual farm in Międzyrzec Podlaski, Poland. The effect of two factors was studied. The first-order factor was two varieties of Helianthus tuberosus: 'Albik' and 'Rubik', and the second-order variants of biostimulant application: Kaishi, Maral, Nutrigreen AD, and Vanadoo. The effectiveness of the application of individual biostimulants in the cultivation of two varieties of Jerusalem artichoke was compared with the control object (without the application of biostimulants). The variety 'Rubik' accumulated the most phosphorus, calcium and potassium and the variety 'Albik' accumulated magnesium. Biostimulants increased the concentration of phosphorus, magnesium and calcium, and decreased potassium compared to topinambur tubers harvested from the control object. Macronutrient content in tubers of the tested varieties Helianthus tuberosus can be ranked in descending order: potassium > phosphorus > calcium> magnesium. The cultivar 'Albik' was characterized by the highest macro-nutrient uptake capacity. The application of biostimulants increased the uptake of phosphorus, magnesium, calcium and potassium compared to the control. The content and uptake of phosphorus, calcium, magnesium and potassium were influenced by climatic conditions.