Three muscadine grape genotypes (Muscadinia rotundifolia (Michx.) Small) were evaluated for their metabolite profiling and antioxidant activities at different berry developmental stages. A total of 329 metabolites were identified using UPLC-TOF-MS analysis (Ultimate 3000LC combined with Q Exactive MS and screened with ESI-MS) in muscadine genotypes throughout different developmental stages. Untargeted metabolomics study revealed the dominant chemical groups as amino acids, organic acids, sugars, and phenolics. Principal component analysis indicated that developmental stages rather than genotypes could explain the variations among the metabolic profiles of muscadine berries. For instance, catechin, epicatechin-3-gallate, and gallic acid were more accumulated in ripening seeds (RIP-S). However, tartaric acid and malonic acid were more abundant during the fruit-set (FS) stage, and malic acid was more abundant in the veraison (V) stage. The variable importance in the projection (VIP > 0.5) in partial least-squares–discriminant analysis described 27 biomarker compounds, representing the muscadine berry metabolome profiles. A heatmap of Pearson’s correlation analysis between the 27 biomarker compounds and antioxidant activities was able to identify nine antioxidant determinants; among them, gallic acid, 4-acetamidobutanoic acid, trehalose, catechine, and epicatechin-3-gallate displayed the highest correlations with different types of antioxidant activities. For instance, DPPH and FRAP conferred a similar antioxidant activity pattern and were highly correlated with gallic acid and 4-acetamidobutanoic acid. This comprehensive study of the metabolomics and antioxidant activities of muscadine berries at different developmental stages is of great reference value for the plant, food, pharmaceutical, and nutraceutical sectors.