A semi-quantitative Real-Time PCR strategy was developed to identify potential indicator organisms for anastomotic leakage in peritoneal drainage fluid, Escherichia coli and Enterococcus faecalis. The analytical performance of the amplification method was validated with 10 culture-positive and 7 culture-negative peritoneal drain fluid samples, obtained from 9 different patients with a colorectal anastomosis. Real-Time PCR results were fully concordant with the microbiological culture results. However, among the culture-negative samples, four false-positive RT-PCR results were found. All false-positives originated from a single patient with a surgical site infection. This may indicate an elevated sensitivity of the RT-PCR method. The results showed that the semi-quantitative RT-PCR method has a clear potential to be useful as a powerful tool in early detection of anastomotic leakage.