Active biomonitoring is used to assess environmental pollution of elements such as heavy metals by indicator species such as mosses. They are used, among others, in urbanized areas where no indicator species are found. In such study areas, mosses collected from sites considered to be ecologically clean shall be exposed. In this context, it is very important to prepare the mosses properly before the exposure, so that the information received about the condition of the environment is reliable. In 2018, studies were conducted in the forested areas of southern Poland—in Opolskie Province. Pleurozium schreberi mosses were used in these studies. Atomic absorption spectrometry with flame atomiser (F-AAS) was used to determine the concentrations of Mn, Fe, Ni, Cu, Zn and Pb present. The aim was to study the influence of preparation methodology on Pleurozium schreberi moss samples prior to use in active biomonitoring studies. Four different methodologies were tested across four different sample locations (with varying levels of pollution). The results of the research were analysed and the coefficient of variation (CV) was determined. The value of the CV is influenced, among other things, by the location of the particular sample and the level of pollution by, for example heavy metals, in the moss. The research conducted proves that of the four methods used to prepare mosses for later exposure in active biomonitoring, the best method is averaging with simultaneous conditioning of mosses in demineralised water. This treatment causes the CV coefficient to fall below 10% for most of the metals determined in the moss samples. It has also been shown that maintaining moss collection methodology in accordance with ICP Vegetation standards (open/wooded area—tree canopy) also has a significant impact on the result obtained. Statistical analysis confirmed (Wilcoxon test) that the method of processing the mosses significantly influenced the results obtained. Thanks to the appropriate preparation of the mosses before exposition, they can be used in active biomonitoring of, for example, urban areas.