This study aims to identification of phytochemical profile of Apis mellifera propolis and explore potential of its anti‐diabetic activity through inhibition on α‐amylase (α‐AE), α‐glucosidase(α‐GE) and finally identified the novel antidiabetic compounds from propolis. Apis mellifera propolis extract (AMPE) exhibited elevated polyphenol 33.26 ± 0.17 (mg GAE/g) and flavonoid (15.45 ± 0.13 mg RE/g), while its also indicated moderate strong antioxidant activity (EC50 793.09 ±1.94 µg/ml). This study found that AMPE displayed promising α‐AE and α‐GE inhibition through in vitro study. Based on LC‐MS/MS screening, 18 unique AMPE compounds were identified, majorly belonging to anthraquinone and flavonoid compounds. In silico study determined that 8 compounds of AMPE compounds exhibited strong binding to α‐AE, interacting to catalytic residue of ASP197. Moreover, 2 compounds exhibit potential inhibition of α‐AG, by interacting to crucial amino acids of ARG315, ASP352, and ASP69. Finally, we suggested 2,7‐Dihydroxy‐1‐(p‐hydroxybenzyl)‐4‐methoxy‐9,10‐dihydrophenanthrene and 3(3‐(3,4‐Dihydroxybenzyl)‐7‐hydroxychroman‐4‐one as novel inhibitors of α‐AE and α‐GE. Notably, these compounds were initially discovered in Apis mellifera propolis, and molecular dynamic analysis confirmed their stable binding with both enzymes over 100 ns simulations. In vivo acute toxicity test reveals AMPE as a practically non‐toxic product with LD50 value of 16050mg/kg.