Manilkara hexandra (Roxb.) Dubard is a large evergreen tree species widely distributed throughout India and other countries and belongs to family Sapotaceae. Conventionally, the entire plant has been used for the treatment of various diseases. The present study has been carried out for identification of its bioactive constituents by high resolution-liquid chromatography-quadrupole-time-of-flight-mass spectrometry (HR-LC-QTOF-MS) and evaluation of in vitro antioxidant and in vitro antidiabetic activity of the methanolic extract of the seeds of the plant. Twenty-eight bioactive compounds from the methanolic extract of Manilkara hexandra seeds (MEMH) were identified using HR-LC-QTOF-MS. In vitro antioxidant activity of MEMH was evaluated using 1,1-diphenyl-2-picryl-hydrazyl, nitric oxide, superoxide, and hydroxyl radical scavenging assays and the IC50 values for MEMH were found to be similar to standard drug. In vitro antidiabetic activity was also evaluated using anti α-amylase and anti-α-glucosidase assays and the IC50 values for MEMH were found to be good as compare to standard drugs. The compounds identified by HR-LC-MS partly explain the various therapeutic effects of the seeds of the plant. Thus, it can be concluded that MEMH is rich in bioactive phytochemicals and possesses potential antidiabetic and antioxidant activity.