The incidence of Pneumocystis jirovecii pneumonia (PJP) is increasing. 108 patients were analysed retrospectively at the Wuhan Union Hospital. The patients were classified into the PJP group or the P. jirovecii colonisation (PJC) group based on clinical diagnosis. Clinical data included demographics, laboratory examinations, treatment, and outcomes. A notable difference in the fungal load was seen between two groups, with median reads of 3215.79 vs. 5.61 in two groups, respectively (P<0.001). The optimal threshold value for discriminating P. jirovecii infection between colonisation for mNGS was six, and serum (1,3)-β-D-glucan (BDG) was 47.6 pg/mL. Besides, the positive detection rate of mNGS for co-pathogens in PJP patients was significantly higher than that of culture (88.16% vs. 22.37%, P<0.0001). Epstein-Barr virus and cytomegalovirus were the most common pathogens of co-infection in PJP patients. The antibiotic therapy in PJP patients was adjusted according to the mNGS results, of which seventeen (22.37%) were downgraded, 38 (50.0%) patients were upgraded, and 21 (27.63%) were unchanged. And almost all patients showed significant improvement in C-reactive protein. mNGS is a promising and valuable technique with good performance for differentiating P. jirovecii infection and colonisation, the detection of pathogens, and antibiotic treatment.