Background <p><i>Pneumocystis jirovecii</i> pneumonia (PJP) is a life-threatening opportunistic infection. Colonization is prevalent but cannot be reliably distinguished from active infection by conventional methods. Metagenomic next-generation sequencing (mNGS) is a promising diagnostic tool, but the value of blood mNGS for diagnosis, microbial community comparison, and outcome-related associations in PJP remains unclear.</p> Methods <p>We analyzed 73 suspected PJP patients with paired BALF and blood mNGS. Using strict diagnostic criteria, patients were classified as: PJP (<i>n</i> = 50) and <i>P. jirovecii</i> colonization (PJC, <i>n</i> = 23). Bioinformatic analyses compared compartment-specific microbiota. BALF-blood concordance and associations between <i>P. jirovecii</i> load and outcomes were evaluated.</p> Results <p>BALF showed higher α-diversity than blood (both Shannon and Simpson, <i>P</i> &lt; 0.001), whereas β-diversity showed no compartmental segregation. BALF identified 216 species versus 43 in blood; however, the top-10 species were concordantly ranked (90% concordance). Blood mNGS distinguished PJP from PJC with an AUC of 0.80 (specificity 95.7%, sensitivity 62.0% at RPM &gt; 4.8), outperforming BALF mNGS (AUC 0.76), blood PCR (AUC 0.64) and BALF PCR (AUC 0.73). Gram-negative bacteria accounted for a large proportion of blood taxa (75% of top 20 taxa), while BALF showed additional fungal taxa including <i>Aspergillus fumigatus</i>. LEfSe identified matrix-specific taxa: oral commensals in PJC-BALF. Blood <i>P. jirovecii</i> load correlated positively with LDH (<i>r</i> = 0.34, <i>P</i> = 0.0035), CRP (<i>r</i> = 0.34, <i>P</i> = 0.0031), and BDG (<i>r</i> = 0.26, <i>P</i> = 0.025), and was higher in non-survivors (<i>P</i> &lt; 0.05).</p> Conclusion <p>Blood mNGS may serve as a non-invasive, highly specific complementary tool for PJP diagnosis and broader microbiological assessment.</p>