Background: Various reasons accounted for the infection of infants kept at Neonatal Intensive Care Unit (NICU). Expressed breast milk may be regarded as a source of infection in infants. This study investigated the source of bacterial contamination and the impact of educational interventions on the contamination of mothers' expressed breast milk (EBM) level whose infants are hospitalized at NICU., Methods: Fifteen mothers used to express breast milking their infants admitted at NICU, involved in this study was conducted during October 2011-March 2012. Samples taken from hands, breast, pumps, breast milk, and milk storage containers and therefore 244 samples were prepared by sterile cotton swab and cultured on Blood Agar and EBM. After presenting enough training to mothers, cultures of the same positions were carried out again. Only those samples proved infected that number of their bacterial colonies exceeded 104 cfu / ml or even there was a growth of pathogenic organism., Results: Before intervention 80% of mothers had infected by at least one sample that reduced to 36% after the intervention. Before intervention 25.4% of samples were contaminated; however after intervention, it reduced to 8.2%. The main source of contamination was milk containers and pumps; moreover, Pseudomonas, E-coli, and Klebsiella were among the most common bacteria of samples' contamination., Conclusion: The possibility of EBM contamination was relatively high but educational interventions might reduce the risk of prevalence.