Fresh pine needles were collected and extracted with 95% methanol and the extract was concentrated to determine its antimicrobial activity. The methanol extract had a considerable inhibitory effect on the tested bacteria, such as Esherichia coli 0157;H7, Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus. The methanol extract of pine needles was further fractionated to chloroform, ethylacetate, butanal, and water fractions. Among these four fractions, the butanol and water fractions, which showed a relatively strong inhibitory effect on all of the tested bacteria, were purified and the minimum ingibitory concentration (MIC) was determined for each microorganism. The MIC raged between 25 mg/ml and 45 mg/ml depending on the microorganism. the purified active fractions were applied to sterilized milk as a model food system to define the antimicrobial effectiveness and it was found that the antimicrobial activities in the water fractions were stronger than those in the butanol fractions.