Pumpkin flowers, in their composition, contain many bioactive ingredients that have a beneficial effect on the human body. The aim of the research was to evaluate the antioxidant activity and chemical composition of flowers of various species and varieties of pumpkins: Amazonka, Ambar, Atlantic Giant, Bambino (Cucurbita maxima L.), Butternut, Muscade de Provence, Rouge vif d’Etampes (Cucurbita moschata Duch.), and Miranda (Cucurbia pepo L.). The flowers came from flowering pumpkin shoots, grown in Poland (Krakow). The total polyphenols, carotenoids, total sugar contents, antioxidant activity, and fatty acid composition were determined. The content of dry matter, protein, ash, fat, and selected minerals were also determined. Pumpkin flowers of the Atlantic Giant variety were characterized by the highest content of total polyphenols and sugars and antioxidant activity. They also showed the highest percentage of n-myristic acid (C14:0) and docosanoic acid (C22:0). The energy value of fresh pumpkin flowers, of all varieties, was low and averaged 22 kcal/100 g. Fresh pumpkin flowers are a significant source of iron, covering 60–80% of the EAR standard for adults (Atlantic Giant and Bambino varieties).