Type 2 diabetes (T2D) is a major health problem worldwide. In this condition, the organism can produce insulin but becomes resistant to it; thus the insulin is ineffective. High blood glucose levels are a result of insulin resistance and insulin deficiency; they produce diabetes-associated complications such as kidney failure, blindness, cardiovascular disease, and lower-limb amputation. In Guatemala, there were over 752.700 cases of the disease in 2017 with prevalence of 8.4 (IDF, 2017). The use of plants for medicinal purposes has been practiced in the country since pre-Hispanic times. Among the Cakchiquels, the aerial parts of Hamelia patens Jacq., Neurolaena lobata (L.) R.Br. ex Cass., and Solanum americanum Mill. and the cortex of Croton guatemalensis Lotsy are used to treat type 2 diabetes. The aim of the present study was to confirm the hypoglycemic effect of the plants under normal conditions and under maltose and sucrose tolerance tests, as well as to test the activity of the plant extracts in vitro against alpha-glucosidases types I and II. In agreement with the traditional usage of the plants, in normal conditions without a sugar load, the extracts produced a statistically significant hypoglycemic effect similar to the control drug glibenclamide. When the sugar load was maltose, only Croton and Solanum produced a statistically significant (p < 0.05) hypoglycemic effect compared to the control drug, but when the sugar was sucrose, Croton and Hamelia produced a statistically significant effect (p < 0.05) beginning at 30 min compared to the control group, while Solanum did so at 60 min and Neurolaena at 90 min. In vitro assays showed that the extracts inhibited yeast alpha-glucosidases but not the rat intestinal ones. Of the tested plants, Croton exert an effect both under sugars' tests and under a normal tolerance test; these results suggest the potential use of this plant. The results presented here provided evidence based on the use of these plants as hypoglycemic agents in the treatment of type 2 diabetes.