Diabetes mellitus remains a global public health problem associated with many complications. Acacia nilotica has been used in local management of diabetes and demonstrated anti-diabetic activities in animal model. Drosophila melanogaster (fruit-fly) has been used as a model for investigating pharmacological activities of natural products because of its genetic resemblance to human genes. This study aims at evaluating the anti-diabetic activities of aqueous leaves extract of Acacia nilotica in HSD-induced diabetic Drosophila melanogaster model. The results showed that flies treated with 0.250 g/mL and 0.500 g/mL of the extract demostrated significant (p < 0.05) increase in locomotor performance compared with diabetic control. The aqueous leaves extract of Acacia nilotica demonstrated significant (p < 0.05) decrease in glucose, glycogen, trehalose and triglycerides levels in the treated flies compared with diabetic control. In comparison with diabetic control, the extract exhibited significant (p < 0.05) decrease in MDA level and increase in SOD, CAT and GSH levels in treated flies. The aqueous leaves extract of Acacia nilotica demonstrates significant anti-diabetic activity in Drosophila melanogaster model validating its anti-diabetic effect in animal model and its use in the local management of diabetes.