Abstract: Introduction: Due to multiple drug resistance against antimicrobial drugs, investigations to discovering new antibacterial compounds from natural sources have been designated. This study was carried out to evaluate the antimicrobial and antifungal activity of the extract and fractions of Euphorbiasplendida Mobayyen. Materials and Methods: Aerial parts of the E.splendida were collected from Arak (Markazi province) and extracted by methanol using maceration method. Crude methanolic extract (CME) was fractionated by liquid-liquid fractionation method using hexane, chloroform, and water. The antibacterial and antifungal activities of the extract and fractions were studied against six bacterial strains and four fungi using minimum inhibitory concentration (MIC) microplate and agar diffusion methods. Total flavonoid content of extract and fractions was determined using AlCl ₃ colorimetric assay. Results: Total extract and fractions of E.splendida, showed a significant antimicrobial effect against tested microorganism. The most effective antibacterial extract was CME with MIC 7.7 mg/ml against Pseudomonasaeruginosa. CME and aqueous fraction showed the best antifungal activity with MIC 3.9 mg/ml against Candidakrusei. Discussion: The findings showed that total methanolic extract of E.splendida possessed antibacterial and antifungal activity. There are some terpenoids and flavonoids isolated from E.splendida which may responsible for the antimicrobial activity. More studies are necessary to determine the active constituent of E.splendida. Conclusion: According to the present study, the aerial parts of Euphorbia splendida showed antibacterial and antifungal activities and can be the basis for further investigation in the potential discovery of new natural antimicrobial compounds.