Sunflower (Helianthus annuus L) is ranked among the leading high-quality edible oil plant and known to possess notable nutritional, culinary and medicinal benefits. This study was aimed to evaluate the phenolic content and profile of methanolic extract from seeds of new seven Sunflower lines as well as their antioxidant and enzyme inhibition activity. The total polyphenolic and flavonoids contents ranged from 22.29–33.09 mg gallic acid equivalent/g and 1.02–3.34 mg rutin equivalent/g respectively with highest values observed in lines BOH3, APO44 and APO45. All lines showed relatively the same phenolic pattern but differ in levels of metabolites accumulation. Phenolic acids were the main component of the extracts. Chlorogenic acid (2835.53–16594.28 μg/g extract) and caffeic acid (2284.57–4142.32 μg/g extract) were relatively of highest abundance in all lines except line APO44. (-)-Epicatechin (24.44–25.37 μg/g extract) was the dominant flavonoid in all lines. Also, it worth to mention that pinoresinol (16.85–21.00 μg/g extract), hyperoside (12.82–23.16 μg/g extract) and hesperidin (10.16–19.56 μg/g extract) were reported in Sunflower seeds for the first time. All lines showed considerable antioxidant activity with lines BOH3 or APO43 displayed significantly (p < 0.05) the highest values in different assays. All lines exhibited significant antityrosinase activity in the range 52.94–60.43 mg Kojic acid equivalent/g. They also inhibited the AChE (1.74–2.49 mg GALAE/g) and BChE (1.97–2.92 mg GALAE/g) enzymes but were less effective towards the α-glucosidase and α-amylase enzymes. Furthermore, to understand the relationship between the phytoconstituents and biological activity, principal component analysis was performed and the analysis was also confirmed this fact. In conclusion, the seeds of these new Sunflower lines contained bioactive molecules which are known for their beneficial health effect and might contributed to the observed antioxidant and enzyme inhibition activities.