Fermented Rosa rugosa ‘Dianhong’ petals with brown sugar, a biologically active food popularized in Dali Prefecture, Northwest Yunnan, China, are rich in bioactive compounds, especially polyphenols, exhibiting strong antioxidant activity. This study evaluated their antioxidant activities, total phenolic contents, and concentrations of polyphenols at different fermentation conditions using different assays: DPPH free-radical scavenging activity, Trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), Folin–Ciocalteu assays, and HPLC–MS/MS and HPLC–DAD methods. The results indicated that fermentation significantly increased (p < 0.05) the antioxidant activity and polyphenol concentration of R. rugosa ‘Dianhong’. Furthermore, Saccharomyces rouxii TFR-1 fermentation achieved optimal bioactivity earlier than natural fermentation. Overall, we found that the use of Saccharomyces rouxii (TFR-1) is a more effective strategy for the production of polyphenol-rich fermented R. rugosa ‘Dianhong’ petals with brown sugar compared to natural fermentation.
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