In the last few decades, the polymerase chain reaction (PCR) has become one of the most powerful molecular biological tools. However, the PCR is an enzymatic reaction and therefore sensitive to inhibitors which may occur in drinking water samples. In this work, the possible inhibition effect of chlorine, humic acids, and iron for real-time PCR (qPCR) efficiency was studied and the environmental sample from drinking water treatment system before iron removal was selected and analysed. The results demonstrated that the highest concentrations of humic acids (5 and 1 mg l-1) and iron (4 mg l-1) inhibited the PCR reaction while no effect of chlorine was observed. The analysis of the environmental sample with spiked Escherichia coli cells demonstrated reduction efficiency of the average threshold cycle (Ct) values compared with control dilution series determining the possible inhibition for qPCR assay.