This study employed in vitro and in silico techniques to screen isolated pregnane phytochemicals (P1-P4), as well as their source pregnane-rich chromatographic fractions, which were derived from Gongronema latifolium Benth (GLB), for their inhibitory potentials against α-amylase, α-glucosidase, and sucrase enzyme activities. The results show that, most pregnane-rich chromatographic fractions and isolated compounds exhibited good inhibitory activities against α-amylase, and α-glucosidase enzymes. Among the isolated compounds, marsectohexol (P2) is the most favourable structure that showed inhibition against α-amylase (IC 50 = 3.712 µg/mL) when compared with the reference inhibitor, acarbose ((IC 50 = 15.418 µg/mL). Based on negative and low value of ∆G (Gibb's free energy), docking scores revealed that, marsectohexol has the highest binding affinity (− 8.8 kcal/mol) to human pancreatic α-amylase (HPA) as compared to the standard acarbose (− 8.1 kcal/mol). Marsectohexol and the other phytocompounds were also found to dock into the active site of HPA; interacting with key catalytic amino acid residues (ASP197, GLU233 and ASP300), which are known to involve in the nucleophilic, and the acid/base catalysis of the enzyme. Although, the pregnane phytocompounds have considerable high affinity binding to the human lysosomal α-glucosidase, they interacted with pockets other than the active site. In conclusion, at least, marsectohexol and three other constituent pregnane phytochemicals of GLB are potential inhibitors of the carbohydrate-hydrolyzing enzymes. These compounds may account for the anti-hyperglyceamic and antidiabetic potentials of this food herb.