Background <p>Accurate and rapid pathogen identification is crucial for the effective treatment of peritoneal dialysis-associated peritonitis (PDAP). Targeted next-generation sequencing (tNGS) has gained popularity for detecting infectious diseases due to its defined pathogen targeting and enhanced detection efficiency. However, its application in the etiological diagnosis of PDAD has not been fully investigated.</p> Methods <p>This prospective study enrolled 35 PDAP patients from November 2023 to January 2025. Both tNGS and traditional microbial culture (TMC) were performed in the peritoneal dialysate simultaneously, with TMC as the reference standard. Discrepant results were adjudicated by clinicians with priority given to TMC.</p> Results <p>tNGS achieved a significantly higher pathogen detection rate (PDR) than TMC (82.9% vs. 48.6%, P = 0.008) and detected 10 polymicrobial infections, whereas TMC identified none. Beyond common pathogens, tNGS also detected rare ones, including <i>Human herpesvirus</i> and the <i>Mycobacterium tuberculosis complex</i>. tNGS also had a significantly shorter detection time than TMC (22.63 h vs. 66.37 h, P &lt; 0.001). Among the patients with prior antibiotic exposure (54.3%), tNGS maintained a higher PDR than that of TMC (89.5% vs. 26.3%, P &lt; 0.001).</p> Conclusions <p>tNGS showed a significantly higher PDR and a shorter detecting time than TMC in patients with PDAP, particularly in those with prior antibiotic exposure. Additionally, this method enables the identification of rare pathogens that are undetectable by TMC. As a complementary tool to conventional methods, combining tNGS with TMC may enable more comprehensive and rapid pathogen diagnosis, thereby improving diagnostic accuracy and patient prognosis.</p> Trial registration <p>This study was registered in the China Clinical Trials Registry (ChiCTR2300078924).</p>