This study aimed to investigate the effect of the maturation process of sweet marjoram (Origanum majorana L.) on essential oil composition, the phenolic profile of ethanolic extract and their antioxidant capacities. The essential oil composition was studied at three stages of maturity by GC–MS. Thirty compounds were detected representing 100% of the total essential oil. p-Menth-1-en-4-ol was the major compound (37.15–76.94%) followed by cyclohexanol-3,3,5 trimethyl (5.41–15.99%) and α-terpineol (0.94–11.34%). During the maturation process, an accumulation of oxygenated monoterpenes was observed. The phenolic composition was studied using matrix-assisted laser desorption/ionization time of flight. The analysis showed the presence of short flavonoid monomers at all stages of maturation. The antioxidant capacity of ethanolic extracts and essential oils was evaluated using the DPPH assay, iron chelating power and reducing power assay. The highest phenolic content and antioxidant capacity were found at flowering stage. These findings on essential oil composition, phenolic profile and antioxidant capacity of O. majorana at three different stages of development provide more information on how these secondary metabolites are accumulated.