Noni seed oil (NSO), a potentially edible oil rich in bioactive compounds, requires refining before consumption. However, this process may alter its chemical composition and functional properties. This study employed foodomics analysis and in vitro activity assays to evaluate the effects of refining on NSO quality, including chemical characteristics, lipid profiles, volatile compounds, phenolic compounds, and anti-inflammatory properties. Refining significantly altered the fatty acid composition of polar lipids, while exerting minimal impact on that of total lipids. The degumming step reduced phytosterols and phenolic compounds, whereas decolorization decreased tocopherol levels. Scopoletin and kaempferol were the predominant polyphenols, with scopoletin being more susceptible to refining. Degumming altered glycerophospholipid and saccharolipid profiles, while deacidification influenced sphingolipid and fatty acyl compositions. Decolorization primarily modified triacylglycerol profiles. Among the 29 volatile compounds identified, (E, E)-2,4-decadienal, ethyl caprylate, ethyl hexanoate, hexanal, and hexyl caprylate were key differential volatiles. Although NSO exhibited potential anti-inflammatory activity, this was notably diminished by the refining process, particularly during deodorization. These findings provide valuable insights for NSO development and quality control.