Jatropha dioica has traditionally been used to treat various health problems. It can be a potential source of bioactive compounds with applications in food, health, and pharmaceuticals. The aim of this study is to investigate the antibacterial effect of J. dioica extracts using UAE- and MAE-assisted extraction methods against pathogenic bacteria in addition to nutritional, phytochemical, and HPLC analysis. The J. dioica extract was screened for nutritional and phytochemical content, and was chemically characterized using HPLC-MS analysis for the presence of compounds. Using ultrasound-assisted extraction (UAE) and maceration-assisted extraction (MAE), the extracts were evaluated for their antibacterial activity against bacterial pathogens using minimum inhibitory concentration (MIC). J. dioica shows a high fiber content (50.56 ± 4.79%) and a large amount of flavonoids, polyphenols, and saponin. Chemical compounds including 3-feruloylquinic acid, caffeic acid 4-o-glucoside, 3,4-dhpea-ea, secoisolariciresinol, caffeic acid ethyl ester, p-coumaroyl malic acid, protocatechuic acid 4-o-glucoside, and 3,7-dimethylquercetin were detected in J. dioica. J. dioica showed potential antibacterial activity against S. aureus with an MIC of 8.33±1.6 mg/mL and E. coli with an MIC of 5.83±2.2 mg/mL. We concluded that J. dioica is a rich source of nutritional, phytochemical, and polyphenolic compounds that promote its biological effectiveness. J. dioica extract had the potential for complete inhibition of E. coli and S. aureus at a low MIC value.