Bronchoalveolar lavage fluid (BALF) is the key sample type for diagnosing Pneumocystis jirovecii pneumonia, with quantitative PCR (qPCR) providing high sensitivity and specificity. However, sample processing varies considerably between laboratories, and optimal nucleic acid extraction method for BALF remains undetermined. This retrospective multicenter study, conducted in 12 centers as part of the fungal PCR initiative, assessed the efficacy of P. jirovecii detection by qPCR in different BALF fractions, including whole (WHO), pellet (PEL), and supernatant (SUP). Samples that were P. jirovecii-qPCR-positive during routine testing were divided into the three predefined fractions prior to nucleic acid extraction and qPCR, comparing detection rates and quantification cycle (Cq) values. Out of 113 P. jirovecii-qPCR-positive BALF samples, 339 qPCR measurements were analyzed. The PEL fraction demonstrated a similar detection rate to the WHO fraction, with positivity rates of 92.9% and 88.5%, respectively. The SUP fraction showed a lower positivity rate of 71.7%, dropping to 47% for high Cq values (Cq >
35). Quantitative analysis showed that the SUP fraction consistently yielded higher Cq values, trailing by 3.05 cycles compared to WHO, while PEL showed a smaller deviation (0.49 cycles), confirming its efficiency in retaining P. jirovecii genetic material for qPCR detection. The study concludes that the SUP fraction is suboptimal for P. jirovecii detection due to higher Cq values, suggesting lower fungal loads. The PEL and WHO fractions are comparable, suggesting that the PEL is a viable alternative, permitting the concentration of larger BALF volumes to levels that can be extracted across a range of platforms.