Abstract: This study investigated the regulatory effects of inoculation timing on the quality of navel orange wine in mixed-culture fermentation. Fermentation protocols were established using Saccharomyces cerevisiae and non-Saccharomyces cerevisiae under four modalities: simultaneous co-inoculation, non-Saccharomyces cerevisiae pre-inoculation, non-Saccharomyces cerevisiae delayed inoculation, and Saccharomyces cerevisiae mono-culture fermentation. Characteristic components and antioxidant activity were systematically analyzed via gas chromatography-mass spectrometry and ultraviolet spectrophotometry. Results demonstrated significant quality modulation by inoculation timing. Mixed-culture fermentation generated 24 additional volatile compounds versus mono-culture. The pre-inoculation group exhibited peak levels of total alcohols at 1500.09 mg/L and total esters at 198.43 mg/L. Total phenolic content reached 753.93 mg GAE/L in the pre-inoculation group, while total flavonoids peaked at 138.30 mg RE/L under simultaneous inoculation. Antioxidant capacity was significantly enhanced in the pre-inoculation group, demonstrating IC₅₀ values of 0.0483 mg/mL for DPPH radical scavenging, 0.071 mg/mL for hydroxyl radical scavenging, and 0.152 mg/mL for ferricyanide reducing power. Sensory evaluation favored the delayed inoculation (DC) and simultaneous inoculation groups (SC), with respective composite scores of 82.13 and 80.13. In conclusion, inoculation timing critically governs flavor quality, functional components, and antioxidant attributes in mixed-culture fermented navel orange wine. Systematic comparison across fermentation modes elucidated yeast interaction mechanisms and quality formation patterns, providing theoretical and empirical foundations for process optimization. This research facilitates value enhancement in citrus wine products and fosters sustainable development in navel orange processing industries.