High temperature (HT) causes physiological and biochemical changes in plants, which may influence their nutritional potential. This study aimed to evaluate the nutritional value of broccoli seedlings grown at HT on the level of phytochemicals, macro- and microelements, antioxidant capacity, and their extracts’ in vitro cytotoxicity. Total phenols, soluble sugars, carotenoids, quercetin, sinapic, ferulic, p-coumaric, and gallic acid were induced by HT. Contrarily, total flavonoids, flavonols, phenolic acids, hydroxycinnamic acids, proteins, glucosinolates, chlorophyll a and b, and porphyrins were reduced. Minerals As, Co, Cr, Hg, K, Na, Ni, Pb, Se, and Sn increased at HT, while Ca, Cd, Cu, Mg, Mn, and P decreased. ABTS, FRAP, and β-carotene bleaching assay showed higher antioxidant potential of seedlings grown at HT, while DPPH showed the opposite. Hepatocellular carcinoma cells were the most sensitive toward broccoli seedling extracts. The significant difference between control and HT-grown broccoli seedling extracts was recorded in mouse embryonal fibroblasts and colorectal carcinoma cells. These results show that the temperature of seedling growth is a critical factor for their nutritional value and the biological effects of their extracts and should definitely be taken into account when growing seedlings for food purposes.