This study aims to develop a technical itinerary for Mg biofortification of two tomato varieties (H1534 and H9205) during the 2018 production cycle. In this framework, plant biofortification was promoted after planting and during the respective production cycle throughout six leaf applications with four different treatments of magnesium sulfate. At harvest, the accumulation of Mg and the interactions with others chemical elements in tomato tissues were studied, as well as the biofortification index. The content of Mg increased 90% and 78.8%, in H1534 and H9205 varieties, respectively, comparatively with controls, as seen by μ-EDXRF M4 Tornado ™ system. Using scanning electron microscopy, with X-ray energy dispersive spectroscopy (SEM-EDS), it was possible to identify in which area of the tomato tissue the Mg predominates. Mg is a micronutrient involved in protein synthesis, muscle and nerve functions, blood glucose control and blood pressure regulation. In human body almost 53% of magnesium (Mg) is involved in the development and maintenance of bone and other calcified tissues. In addition, Mg deficiency is uncommon in humans because of the kidney urinary excretion limits. However, there are some groups at risk of Mg inadequacy, people with gastrointestinal diseases, type 2 diabetes, chronic alcoholism. Magnesium biofortification is a strategy that boosts nutrient enhancement in food crops and can increase nutrient uptake and accumulation in the human body. Considering that biofortification with Mg consists in the development of a functional product, it is concluded that it’s possible to develop a new product such as biofortified tomato pulp.