Spinach (Spinacia oleracea L. SO) leaves represent an important dietary source, have high nutritional value and antimicrobial properties. Spinach leaves have been used in the treatment of human diseases since ancient times. Here, the aim of this study was to evaluate the antimicrobial and antifungal activities of ethanolic extract of Spinach oleracea leaves by determining the minimum inhibitory concentrations (MICs) using well diffusion method against bacterial species Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus and Klebseilla pneumonia and fungal species Aspergillus fumigatus, Aspergillus niger, Candida albicans and Fusarium oxysporum. We evaluated from present data that ethanolic extract of root showed zone of inhibition ranges from 6 mm to 21 mm, ethanolic extract of stem showed zone of inhibition ranges from 8 mm to 21 mm and ethanolic extract of leaf showed zone of inhibition ranges from 9 mm to 22 mm from concentration 25 mg/ml to 100mg/ml. Leaf extract has high antibacterial and antifungal activity against bacterial and fungal species while root extract has low antibacterial and antifungal activity against bacterial and fungal species. Activity of plant extract was increased by the increasing concentration of extracts. Very low zone of inhibition was found at concentration 25 mg/1 ml DMSO which ranges from 6 mm to 14 mm while very high zone of inhibition was found at concentration 100 mg/ml which ranges from 6 mm to 22 mm. So, ethanolic extracts of Spinach oleraceae has good efficiency against bacterial and fungal species.