The aim of the present study was to compare the effectiveness of selected DNA extraction methods for the detection of Rhodococcus equi from tracheobronchial wash fluid by PCR. Three methods of nucleic acid extraction were evaluated, based mainly on the activity of proteolytic enzymes. A commercial kit for isolation and purification of bacterial DNA was also used in the study. In one procedure, an additional component, the cationic detergent CTAB, was used. It has been found that the traditional enzyme digestion methods used with the tracheobronchial wash fluid are more suitable to prepare DNA matrix for PCR comparing with commercial DNA isolation kit. Minimum numbers of bacteria detected with the use of traditional enzyme digestion methods and commercial kit were 100 and 500 cells, respectively. Based on the results of the study we can recommend the enzymatic digestion method along with CTAB as an additional component.