Plant phytochemical accumulation is influenced by various external factors that change with the seasons (e.g., harvesting time). Atractylodes japonica, an important medicinal plant rich in bioactive compounds, is used to treat several human diseases. We analyzed the influence of harvesting time on phenolic compound concentration and antioxidant activity of A. japonica roots. We investigated the correlation between phenolic compound and minerals contents and antioxidant activity in different harvests. Total phenolic and flavonoid contents varied significantly with the harvesting time. Liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis revealed significant changes in the concentration of various phenolics between harvests. The content of different types of phenolics were significantly higher in the samples collected in October. Among them, chlorogenic acids (133,833.30 µg/g dry weight of root extract) were the most dominant phytochemical compounds detected. Samples harvested in October had higher concentrations of flavonoids, including rutin, orientin, vitexin, and apigenin. Roots harvested in October had a significantly higher (p < 0.05) antioxidant activity than that of those harvested later. Root mineral concentrations also varied with the harvest time. The analysis revealed that macro elements such as Ca ad Mg contents were significantly increased with delaying harvesting time, whereas a different trend was observed for the microelements including Fe, Cu, Al, and As contents in the October harvest. We also found a significant relationship between antioxidant activity and phenolic compound content. The most abundant minerals (Ca, Mg, Mn, Fe, and Al) correlated positively with the antioxidant activity indicating that these elements and compounds may be associated with the A. japonica antioxidant potential. Furthermore, A. japonica root extracts inhibited NIH/3T3 cellular proliferation in a season- and dose-dependent manner. Hence, harvesting time influenced the antioxidant properties and phenolic compound accumulation of A. japonica roots. These results indicate that the harvesting time is essential for obtaining the specific phytochemicals.