Background Ventilator associated pneumonia (VAP) is associated with high mortality and morbidity. Unfortunately, conventional Methods often fail to identify the etiology of VAP and accurate microbial diagnosis remains difficult. As a result, broad-spectrum antimicrobial treatment is frequently used for long durations without clear evidence. We compared the use of nanoporebased metagenomic sequencing to conventional diagnostic Methods in the diagnosis of VAP. Method Patients who underwent bronchoscopy with suspicion of VAP between February 2020 and February 2021 in a medical intensive care unit of a university affiliated hospital were screened for analysis. There were 37 cases in which both the conventional diagnostic Methods and nanopore sequencing were performed using bronchial washing or bronchoalveolar lavage specimens. The nanopore sequencing Results were compared to the conventional diagnostic Methods. Results Among the 37 cases, conventional culture was positive in 12 cases and negative in 25 cases. In culture-positive cases, nanopore sequencing detected the same pathogen in 11 cases (concordance rate 91.7%). Nanopore sequencing did not detect S.maltophilia in 1 case. In culture-negative cases, nanopore sequencing also were negative in 6 cases and upper respiratory tract flora was detected in 5 cases (concordance rate 44%). Additional microbiological diagnosis was possible using nanopore in 14 cases. The additional microorganisms detected were E.faecium, A.baumanni, S,maltophilia, P.jirovecii, E.coli, S.aureus, L.pneumophilia, etc (table 1). Conclusion In patients with clinical suspicion of VAP, nanopore sequencing of bronchoscopic specimens showed high concordance rate with the conventional diagnostic Methods. In culture-negative cases, additional microbiologic diagnosis was also possible using nanopore sequencing.