Background: Pneumocystis pneumonia (PCP) commonly affects immunocompromised individuals, whereas in immunocompetent persons, it occurs relatively rarely, and in most cases the Pneumocystis infection is detected as an asymptomatic colonization. The present study aimed to establish the prevalence of Pneumocystis jirovecii infection in human hosts with different immune status (immunocompromised and immunocompetent), using molecular diagnostic methods, and to compare their diagnostic value with that of classical staining methods. Methods: We used the collected to this moment data from a prospective study on the prevalence of pneumocystosis among the Bulgarian population. Clinical specimens (including throat secretion, induced sputum, tracheal aspirate, and bronchoalveolar lavage collected from 220 patients suspected of PCP (153 immunocompetent and 67 immunocompromised patients) were examined with staining microscopic methods and real-time PCR for detection of P. jirovecii. Results: DNA of the pathogen was detected in 38 (17%) specimens (32 immunocompromised patients and 6 immunocompetent subjects). From all 220 clinical samples examined by staining methods, only in five (2%) P. jirovecii cysts were detected by the Gomori's stain. All patients with PCP were treated with trimethoprim-sulfamethoxazole, but in ten of them (HIV- positive patients) the disease was with fatal outcome. Conclusions: This study is the first for the country including the main available laboratory methods for diagnosis of human pneumocystosis in Bulgaria. Regarding the etiological diagnosis of PCP, in our study the sensitivity of real-time PCR was higher compared to the staining methods. The choice of a method for sample collection and examination has an important role in the efficiency of the laboratory diagnostics.