Background and objective: Candidemia is a one of the most common causes of late-onset sepsis in the neonatal intensive care unit (NICU) and is associated with significant morbidity and mortality. The aim of this work is to evaluate the value of Real-Time PCR for early detection of blood stream Candida albicans infection (candidemia) in NICU. Materials and Methods: The study included 60 newborn admitted in NICU of Al-Zhraa hospital, Al-Azhar University Hospital for girls, Egypt, during the period from January  2010 to January  2011,  who had one or more risk factors for candidemia. A total of 60 blood samples were submitted to testing by blood culture and real-time PCR. 20 cases were diagnosed as candidemia by blood culture method, the other 40 neonates were negative for candidemia  by blood culture method considered as control group. Results: the blood culture was positive in 20/60 samples and Candida albicans was the predominant cause of candidemia 15/20 (75 %) followed by C. tropicalis 4/20 (20 %) and C. glabrata 1/20 (5 %). Real-time PCR for Candida albicans was positive in 14/15 and gave only one false-positive and false-negative result. It had a sensitivity, specificity and accuracy of 93.3 %, 97.8 % and 96.7% respectively. Central venous catheter (CVC) was the most frequent risk factor among candidemic cases 15/20 (75 %), followed by mechanical ventilation (MV), endotracheal tube (ETT) and admission > 10 days in NICU (45 %) for each. Out of 20 patients, 17 had more than one risk factor (Odd Ratio [OR] 22.7; 95%CI 5.31, 96.77; p<0.0001). Conclusion: Real-Time PCR can be applied as a rapid diagnostic method for candidemia and can aid in early antifungal therapy and in minimizing the several drawbacks of candidemia. Identification of risk factors is more important in predicting the neonates who are at risk for candidemia.