Phytochemical investigation on the fruits of Cydonia oblonga Mill., a traditional Uighur medicine, led to the isolation of seven undescribed and nine known megastigmane glycosides. Their structures including absolute configurations were characterized by an extensive analysis of spectroscopic data including HRESIMS and NMR, combined with ECD calculations. Additionally, compounds 1, 2, 4, and 6–16 exhibited anti-inflammatory activity by inhibiting the secretion of cytokines TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharides (LPS) with inhibitory rates of 10.79%–44.58% at 20 μM. Seven undescribed megastigmane glycosides along with nine known analogues were isolated from the fruits of Cydonia oblonga Mill. Several compounds showed anti-inflammatory activity. Display omitted •Sixteen megastigmane glycosides were isolated from Cydonia oblonga.•Seven previously undescribed megastigmane glycosides were included.•Several compounds showed anti-inflammatory activity.