Spirodela polyrhiza is a rapidly growing floating plant with high biomass productivity and the ability to accumulate diverse metabolites, indicating potential applications in the pharmaceutical and cosmetic industries. Most duckweed studies use conventional-density conditions (CDC), in which cultures are harvested before complete surface coverage. In this study, high-density conditions (HDC) were defined as the stage beyond full surface coverage characterized by multilayered mat formation. We investigated biomass accumulation during prolonged cultivation to the HDC stage and analyzed metabolites using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–mass spectrometry (LC–MS). GC–MS detected 52 metabolites, whereas LC–MS detected 23 metabolites, including 20 flavonoids. From CDC to HDC, total dry weight increased by 414%. Relative yields per culture volume also increased for γ-aminobutyric acid (2.20 log₂FC), β-sitosterol (1.70 log₂FC), campesterol (2.45 log₂FC), stigmasterol (2.21 log₂FC), vitexin (2.31 log₂FC), and luteolin 7-O-glucoside (2.42 log₂FC). Thus, S. polyrhiza continued to accumulate biomass during prolonged cultivation to the HDC stage without external elicitors or genetic modification, accompanied by increased relative yields of several metabolites per culture volume. These findings suggest that extending cultivation to the HDC stage may increase biomass and metabolite yields.