Due to wide abuse of antibiotics both in human and livestock use, the advent and spread of multidrug resistant (MDR) pathogens becomes a serious health problem all over the world. Since the development of new antibiotics is at a standstill in pharmaceutical industry, the choice of therapeutic antibiotics is getting narrower. In this study, in an effort to search new antibiotics, the antimicrobial activity of Paenibacillus DY1 isolated from Korean soil was characterized on its growth inhibition spectrum against various health threatening MDR strains, with its stability and chemical structure. Extracellular culture filtrate of Paenibacillus DY1 effectively inhibits the growth of all the tested MDR enteropathogenic Eshcherichia coli, enterohemolytic E. coli, and enterotoxigenic E. coli strains, at a similar level to that on the nonresistant control E. coli strains. It showed significant growth inhibition effect against the causative agents of class one legal communicable disease, MDR Salmonella typhi, MDR Salmonella paratyphi A, food poisoning bacteria, MDR Salmonella typhimurium, and other MDR Salmonella spp. The growth of all of 10 different MDR Shigella spp. strains and 6 different Vibrio spp. strains tested was also inhibited. The antimicrobial activity of Paenibacillus DY1 was well preserved after heat treatment, and was also stable in both alkaline and acidic environment. The antimicrobial activity was partially purified with Diaion HP20 column and TLC. By NMR study, the putative structure of the activity was postulated as an alkane having hydroxyl groups.