As lupin has emerged popularity as dietary protein and nutritional source, our present research was aimed to demonstrate the antidiabetic and organ-protective activities of nine cultivars of Australian sweet lupin seed flours by means of in vitro and in vivo assays accompanied by identification of their bioactive phytocompounds and exploration of underlying mechanisms of their hypoglycemic activity using in silico approach. In vitro α-amylase and α-glucosidase activities inhibition and glucose uptake assays identified Jenabillup seed flours for exhibiting the most potential antidiabetic activity amongst the nine cultivars. In vivo antidiabetic and major organ-protective activities were investigated on streptozotocin-induced hyperglycemia and organ damages in Wister rat model. Along with attenuating hyperglycemia and retreating major organ damages, the biochemical imbalance in cardiac, hepatic and renal markers were well-balanced by Jenabillup seed flours treatment. These activities of lupin seed flours were insignificantly affected by thermal processing. Moreover, in silico investigation of 106 phytochemicals identified by gas chromatography-mass spectroscopy (GC–MS) analysis of the seed flour extracts of nine cultivars revealed that more than 35% of compounds possess moderate to high binding affinity to α-amylase and α-glucosidase enzymes. These bioactive compounds act synergistically to exert potential hypoglycemic activity. Cross-docking and binding energy calculation by molecular mechanics/generalized Born volume integration (MM/GBVI) model suggest actinomycin C2 as a potential inhibitor of both α-amylase and α-glucosidase enzymes. These findings acclaim that Australian sweet lupin seed flours may be considered not only as functional food, but also for further development of effective drugs in pharmaceuticals in the treatment of diabetes mellitus and resultant organ damages.