Palm wine is a traditional alcoholic drink and act as an excellent substrate for microbial growth, which was obtained from the sap of various types of palm trees. The pH and alcoholic content of the fresh palm wine were determined as 7.2 and 0.09%. Yeast, aceticacid and lactic acid bacteria were isolated from fresh palm wine by pour plate technique using suitable selective media by providing optimum growth conditions. The morphological and biochemical characterization identifies the cultures as Saccharomyces, Lactobacillus and Acetobacter sp. Antimicrobial activity of yeast and Lactic acid bacteria was determined by performing diffusion assays. Among the two organisms yeast produced significant antimicrobial activity with 26 mm of inhibition zone diameter against Staphylococcus aureus, and 22 mm of inhibition zone diameter against Pseudomona aureoginosa.