The main goals of this research were the chemical and biological characterization of the bitter melon (Momordica charantia) isolate obtained by traditional (maceration) extraction, as well as optimization of this process using response surface methodology (RSM) and artificial neural networks (ANNs). Experiments were performed using Box–Behnken experimental design on three levels and three variables: extraction temperature (20 °C, 40 °C, and 60 °C), solvent concentration (30%, 50%, and 70%) and extraction time (30, 60, and 90 min). The measurements consisted of 15 randomized runs with 3 replicates in a central point. The antioxidant activity of obtained extracts was determined by the 1,1-diphenyl-2-picrylhydrazyl (DPPH), cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays while chemical characterization was done in terms of the total phenolic content (TPC). The methodology shows positive influence of solvent concentration on all four observed outputs, while temperature showed a negative impact. RSM showed that the optimal extraction conditions were 20 °C, 70% methanol, and an extraction time of 52.2 min. Under these conditions, the TPCs were 20.66 milligrams of gallic acid equivalents (mg GAE/g extract), DPPH 30.22 milligrams of trolox equivalents (mg TE/g extract), CUPRAC 67.78 milligrams of trolox equivalents (mg TE/g extract), and FRAP 45.48 milligrams of trolox equivalents (mg TE/g extract). The neural network coupled with genetic algorithms (ANN-GA) was also used to optimize the conditions for each of the outputs separately. It is anticipated that results reported herein will establish baseline data and also demonstrate that that the present model can be applied in the food and pharmaceutical industries.