Milk, a cornerstone of neonatal nutrition and human health, harbors various components with antioxidant capabilities, including oligosaccharides. In this study, we explored the antioxidant potential of acetylated milk oligosaccharides (A-MOs) and deacetylated oligosaccharides (D-MOs) sourced from Rathi cow milk through assays, with a focus on their comparative efficacy. Concurrently, we evaluated the antioxidant activity of Ascorbic Acid (AA) for comparison. Results revealed significant dynamics within the antioxidant profiles of A-MOs and D-MOs. A-MOs demonstrated concentration-dependent antioxidant activity, with higher concentrations correlating with increased efficacy, while D-MOs displayed a distinct pattern, with varying antioxidant potential across concentrations. Notably, both A-MOs and D-MOs exhibited promising antioxidant activity, suggesting their potential as natural antioxidants. In contrast, AA exhibited a decline in antioxidant activity with increasing concentration, indicating a differing pattern from oligosaccharides. These findings underscore the significance of acetylation in modulating the antioxidant properties of milk oligosaccharides. Further investigation is warranted to elucidate the mechanisms underlying these observations and to explore the practical implications of A-MOs and D-MOs as functional food components or potential therapeutic agents. Overall, our study contributes to the understanding of milk oligosaccharides' antioxidant properties and highlights the potential of acetylated and deacetylated oligosaccharides from Rathi cow milk as valuable sources of natural antioxidants with diverse applications in nutrition and healthcare.