The phospholipids method for the determination of the living cell content in the biofilm was established to study the wastewater biofilm. With the micro-slicing technology, the biofilm was divided into 150 microm-layer for the phospholipids analysis. Results showed the phospholipids were approximately normal distribution along the biofilm depth without no addition of pentachlorophenol (PCP). The absolute values were between (1 246.2 +/- 217.4) microg/L and (527.2 +/- 95.3) microg/L with the maximum occurring at the depth of (0.60 +/- 0.15) microm, indicating the highest concentrations of the living cell in the biofilm. With the PCP concentrations of 5 microg/L, 10 microg/L and 20 microg/L and the exposure times of 2 h, 6 h and 24 h, respectively, the results of the orthogonal experiments showed the value of phospholipids decreased greatly along the biofilm depth only with the exception of 5 microg/L PCP concentration and 2 h exposure time, with a concentration decline by up to 70% especially close to the peak of phospholipids distribution. Results proved that PCP has an adversely influence on the living cells, and the influence can be enhanced with the increase of PCP amount and the exposure time.