Background: Past history indicates that plants were served as an important source of medicine. Otherwise, in developing countries people use medicinal plants against infectious disease because they cannot afford expensive drugs. Due to increasing rate of drug-resistant diseases, there is an urgent need to detect novel antimicrobial compounds from medicinal plants. Objectives: The aim of the present study was to determine Antimicrobial activity of Ephedra pachyclada methanol extract on some enteric Gram-negative bacteria which causes nosocomial infections by agar dilution method. Methods: In this cross-sectional study, in order to examine the antimicrobial effects of Ephedra pachyclada extract on intestinal Gram-negative bacteria, we exposed them to 0/128, 0/25, 0/5, 1, 2, 4 and 8 mg/mL of the extract. Ephedra pachyclada was collected from Jiroft Heights and methanolic extract was prepared with maceration method, during which, 50 gr powder of Ephedra pachyclada was dissolved in 300 mL of 80% methanol. Results: In this study, the antibacterial effects of Ephedra pachyclada extract on Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli (PTCC-O157), Escherichia coli (ATCC-25922), Klebsiella pnemoniae, Serratia marcescens was investigated, defining the minimum inhibitory concentration (MIC) by agar dilution method. It has been demonstrated that methanolic extract of Ephedra pachyclada affect intestinal Gram-negative bacteria. Conclusions: The result showed that, Ephedra pachyclada extract has effective antimicrobial ingredients which are cheap and readily available. It can be used for medicinal purposes in the production of antimicrobial drug.