(1,3)-β-D-Glucan (BG) assay is a non-invasive test commonly used in the diagnostic of invasive fungal diseases. Given its high sensitivity, it was suggested that a negative BG result is sufficient for exluding the diagnosis of Pneumocystis pneumonia (PCP). However, suboptimal performance has been described in HIV (Human Immunodeficiency Virus)-negative patients, particularly those with haematological malignancies. We aimed to assess the sensitivity of the BG assay for diagnosing PCP in HIV-negative patients based on their underlying PCP risk factors. We conducted a single-center, retrospective study (2009-2021) enrolling HIV-negative patients diagnosed with PCP and who underwent BG testing. Patients colonized with Pneumocystis jirovecii were included as a control group. In all, 55 PCP patients and 61 colonized patients met the inclusion criteria. Patients were further categorized according to their underlying condition exposing to PCP. Median BG concentration was significantly higher in the PCP group than in the colonization group (500 vs. 31 pg/ml; p < 10−4, Mann-Whitney test) and the BG assay demonstrated a sensitivity of 85% and a specificity of 82% for PCP diagnosis. Notably, sensitivity was significantly higher in non-cancer patients (100%) compared to those with solid cancer (72%) and haematologic cancer (79%) (p < 0.05, Fischer's exact test). These findings strengthen the high performance of BG testing for screening PCP in non-cancer patients, comparable to that observed in HIV-infected individuals. In contrast, they highlight its low reliability in patients with malignancies, emphasizing the importance of considering underlying conditions when interpreting BG results and refining the role of the test in PCP diagnosis.