Sesame seeds are important resources for relieving oxidation stress-related diseases. Although a significant variation in seeds’ antioxidant capability is observed, the underlying biochemical and molecular basis remains elusive. Thus, this study aimed to reveal major seed components and key molecular mechanisms that drive the variability of seeds’ antioxidant activity (AOA) using a panel of 400 sesame accessions. The seeds’ AOA, total flavonoid, and phenolic contents varied from 2.03 to 78.5%, 0.072 to 3.104 mg CAE/g, and 2.717 to 21.98 mg GAE/g, respectively. Analyses revealed that flavonoids and phenolic acids are the main contributors to seeds’ AOA variation, irrespective of seed coat color. LC-MS-based polyphenol profiling of high (HA) and low (LA) antioxidant seeds uncovered 320 differentially accumulated phenolic compounds (DAPs), including 311 up-regulated in HA seeds. Tricin, persicoside, 5,7,4′,5′-tetrahydro-3′,6-dimethoxyflavone, 8-methoxyapigenin, and 6,7,8-tetrahydroxy-5-methoxyflavone were the top five up-regulated in HA. Comparative transcriptome analysis at three seed developmental stages identified 627~2357 DEGs and unveiled that differential regulation of flavonoid biosynthesis, phenylpropanoid biosynthesis, and stilbene biosynthesis were the key underlying mechanisms of seed antioxidant capacity variation. Major differentially regulated phenylpropanoid structural genes and transcription factors were identified. SINPZ0000571 (MYB), SINPZ0401118 (NAC), and SINPZ0500871 (C3H) were the most highly induced TFs in HA. Our findings may enhance quality breeding.