Bread is an ideal food vector for fortification with carotenoids because it is a staple food in many parts of the world, providing an essential source of carbohydrates, proteins, and minerals to millions of people. However, bread alone is not sufficient to meet the nutritional requirements of a balanced diet; because in many parts of the world, individuals suffer from micronutrient deficiencies such as vitamin A deficiency, bread fortification with synthetic or natural provitamin A carotenoids can provide a solution. The fortification of bread with carotenoids of natural origin is an important factor in ensuring consumer acceptance and compliance, hence this study targets a fortified bread obtained through the straight-dough procedure, in which a puree made from Cucurbita maxima Duch. fruits was added. Because carotenoids are heat-sensitive biomolecules, high performance liquid chromatography (HPLC) with photodiode-array detection was used as method of analysis, this being the most appropriate analytical technique for their sensitive and selective analysis. Reversed-phase separations were achieved using a gradient with acetonitrile: water (9: 1) and ethyl acetate, this leading to a good separation of four carotenes (α-carotene, β-carotene, 9Z-β-carotene and 15Z-β-carotene) and eight xanthophylls (neoxanthin, violaxanthin, cucurbitaxanthin A, lutein, zeaxanthin, α-cryptoxanthin, β-cryptoxanthin and 5,6-epoxy-β-carotene). HPLC analysis highlighted that the major carotenoids from fortified bread were lutein, cucurbitaxanthin A and β,β-carotene as well as the stability of the targeted compounds and the change in provitamin A activity as a result of thermal processing during baking; the most stable carotenoids proved to be lutein and cucurbitaxanthin A. By the obtained results, this study offers not only a possible solution for combating vitamin A deficiency but also a true functional food, which can open new market for producers; meanwhile, the behavior of the involved carotenoids can provide relevant information for nutritional studies. Fortified bread may also be of interest to consumers due to its health benefits, since the involved carotenoids have also other effects, such as the antioxidant one and the age-related macular degenerations’ prevention.