β-D-glucan is extensively employed as a supplementary diagnostic tool for Pneumocystis pneumonia (PCP) and typically yields positive results in most cases. We present a case of a 73-year-old woman with a history of rheumatoid arthritis, who was receiving biological agents and was admitted due to pneumonia. Initially, the β-D-glucan test was negative. However, as the disease progressed, it eventually turned positive, leading to the diagnosis of PCP. The patient was treated with corticosteroids and trimethoprim-sulfamethoxazole, resulting in pneumonia resolution. Our findings suggest that repeated assessment of β-D-glucan levels holds diagnostic value in patients without human immunodeficiency virus infection.