Rosemary (Rosmarinus officinalis L.) is a woody, evergreen perennial medicinal plant of the Lamiaceae family that is rich in phenolic, flavonoid, and terpenoid compounds. Rosemary is known for its analgesic, anti-rheumatic, and carminative properties and is traditionally used to alleviate respiratory disorders and epilepsy. The importance of rosemary in the pharmaceutical, cosmetic, and healthcare industries, together with its adaptability to diverse environmental conditions, has attracted considerable research interest. Therefore, this study aimed to evaluate the effect of harvest time on biomass production, phytochemical characteristics, essential oil yield, and essential oil composition of rosemary. This study was conducted as a randomized complete block design (RCBD), with three harvest times (March, June, and July), and five replications during the 2022–2023 growing seasons at a rosemary farm in Gotvand, Khuzestan Province, Iran. The evaluated traits included fresh and dry biomass, total phenolic content, total flavonoid content, antioxidant capacity, essential oil content, essential oil yield, and essential oil composition. Essential oil constituents were identified using GC–MS. Analysis of variance showed that harvest time significantly affected fresh and dry biomass, total flavonoid content, and essential oil content, but had no significant effect on total phenolic content or antioxidant capacity. GC–MS analysis identified 30 compounds, 13 of which were major constituents. Harvesting in July maximized essential oil yield, indicating that this harvest time is optimal for rosemary cultivation under the climatic conditions of Khuzestan Province.