In this investigation, potential inhibitors of α-amylase, one of the key regulatory enzymes in diabetes were characterized from the methanolic extract of Leucas ciliata Benth. (Lamiaceae), a traditional medicinal plant of the Western Ghats, southern India and the ethyl acetate extract of Streptomyces longisporoflavus (JX965948), an endophytic actinomycete isolated from the stem fragments of L. ciliata, by Gas Chromatography and Mass Spectroscopy (GC–MS) technique followed by molecular docking studies. Forty-four compounds were detected in the solvent extracts of the host plant and the endophyte, respectively. These compounds were selected as ligands for the receptor α-amylase in the molecular docking studies using PyRx software (0.8 V) for the inhibition of α-amylase activity. The ligands were ranked based on the binding energies ranging between − 3.1 and − 10.1 kcal/mol. Three ligands from the host plant extract, viz., Topotecan (PNo_7), Cathine (PNo_17) and 2,5-dimethoxy-4-(methylsulfonyl)amphetamine (PNo_18), depicted good binding energies of − 5.2 to − 7.8, respectively, whereas seven compounds from the endophyte extract showed binding energies in the range of − 4.7 to − 10.1, respectively. The standard α-amylase inhibitor Acarbose™ depicted binding energy of − 9.2 kcal/mol. All ligands were subjected to lead-likeliness property using Lipinski’s rule of five. On the basis of the hydrogen bonding interactions with the receptor, and chemoinformatics analysis for drug-likeliness, one ligand, Topotecan (PNo_7) from the host plant was identified as the potential α-amylase inhibitor. This is the first attempt to identify alkaloid and flavonoid compounds as the α-amylase inhibitors from the host plant and its endophyte simultaneously. The molecular docking analyses presented in this study could lead to the development of potent α-amylase inhibitors helpful in the treatment of diabetes. © 2021, King Abdulaziz City for Science and Technology.