<p>Metagenomic next-generation sequencing (mNGS) offers sensitive and rapid pathogen detection and has the added advantage of analyzing chromosomal copy number variations (CNVs) of the host in the same test. This study aimed to evaluate the dual diagnostic performance of mNGS in clinical cases where pulmonary infections and malignancies are difficult to be distinguished. A single-center prospective study was conducted at the First Affiliated Hospital, Zhejiang University School of Medicine. The study recruited patients with lung lesions from October 2021 to October 2022 that required differential diagnosis of infection, malignancy, or other pulmonary diseases. The mNGS was used to detect both pathogens and potential CNVs of the host chromosomes to inform presence of neoplasm, and the results were compared to conventional microbiological tests (CMTs), bronchoalveolar lavage fluid (BALF) cytology, histology, and clinical composite diagnoses. The mNGS demonstrated a significantly higher sensitivity of infection diagnosis (56.5% vs.39.1% for CMTs, P &lt; 0.05). The CNVs analysis showed moderate sensitivity (38.9%) and high specificity (100%) for diagnosing malignancy, which helped determine lung cancer in 4 cases who were initially considered as pneumonia. Combining CNVs analysis with BALF cytology has increased the sensitivity for detecting malignancy from 38.9% to 55.6%. Furthermore, the sensitivity of CNVs analysis was higher (50%) when bronchoscopy directly found positive signs, such as neoplasm or bronchial mucosal infiltration. This study highlights the dual capacity of mNGS to simultaneously detect infections and malignancies. The integration of both pathogen and tumor detection could significantly enhance clinical decision-making, particularly for patients with overlapping symptoms of infection and cancer.</p>