This study systematically evaluated and compared the effects of six extraction methods, namely hot water extraction (HWE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), acid-assisted extraction (CAE), alkali-assisted extraction (AAE), and enzyme-assisted extraction (EAE), on the structural characteristics, in vitro biological activities, and cytotoxicity of polysaccharides from chestnut flowers (CFPs). The results show that CFPs extracted by different extraction methods have significant differences in terms of chemical composition, monosaccharide spectrum, molecular weight distribution, and surface morphology. However, their similar infrared spectra, crystal structures and thermal stabilities indicate that despite the different degrees of degradation, the main structure of CFPs remains basically intact in different extraction methods. It is worth noting that the CFPs produced by HWE have the strongest antioxidant activity (98.5 ± 0.48%, evaluated by the DPPH free radical scavenging assay), while the CFPs produced by EAE have the highest hypoglycemic activity (94.3 ± 0.4%, evaluated by the α-glucosylase inhibition assay). Furthermore, the CFPs of all extraction methods showed biocompatibility. Under the condition of conforming to physiological relevance, the selected cell concentrations all promoted the proliferation of RAW264.7 mouse macrophages, indicating their lack of cytotoxicity. These findings provide a theoretical basis for the selection of CFPs extraction methods with targeted biological activity. Specifically, HWE is recommended for the production of CFPs rich in antioxidants, while EAE is the best choice for preparing CFPs with hypoglycemic properties. This study also lays a foundation for further research on the in vivo biological activity of CFPs.