Due to its high nutritive value, the butternut squash (Cucurbita moschata Duch. ex Poir) has, in the past several years, become an ever more popular vegetable. Many abiotic and biotic factors can influence the quantity of minerals and bioactive substances in the butternut squash, which makes it necessary to analyse the nutritive content of butternut squash cultivars offered on the Croatian market. Contemporary agriculture is seeing a rise in the use of biostimulants, natural substances which increase yield and quality of the cultivated plant and protect it from stress without any negative effect on the environment. In order to research the effect of biostimulants ('Actiwave', 'Bioplex' and 'Megafol') on the quantity of minerals and β-carotene in the fruit of various butternut squash cultivars ('Argenta', 'Futsu' and 'Blue boy') a field experiment was conducted on the experimental field of the Faculty of Agriculture in Zagreb, during the summer-autumn cultivation period of 2015. The highest content of crude proteins (26.72 %), phosphorus (1.24 % P), potassium (4.68 % K), iron (89.29 mg Fe/kg dry weight), zinc (52.42 mg Zn/kg dry weight), manganese (14.28 mg Mn/kg dry weight) and copper (16.60 mg Cu/kg dry weight) were determined in the fruit of the 'Futsu' cultivar. Treating plants with biostimulants had a negative effect on dry weight accumulation and β- carotene synthesis in the butternut squash fruits, since the statistically highest values of these properties (7.77 % and 2725 μg β-carotene/100 g fresh weight) were found on control plots. The 'Futsu' × 'Bioplex' combination resulted in phosphorus, potassium, iron, zinc, manganese, copper and crude protein values comparable to the highest ones. The average phosphorus and potassium quantities in the fresh weight of butternut squash fruits were 54.44 mg P/100 g and 245.59 mg K/100 g, while the average quantities of microelements (iron, zinc, manganese and copper) were 0.404 mg Fe/100 g, 0.254 mg Zn/100 g, 0.064 mg Mn/100 g and 0.078 mg Cu/100 g.