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Rationale & Objective Conventional culture can be insensitive for the detection of rare infections and for the detection of common infections in the setting of recent antibiotic usage. Patients receiving peritoneal dialysis (PD) with suspected peritonitis have a significant proportion of negative conventional cultures. This study examines the utility of metagenomic sequencing of peritoneal effluent cell-free DNA (cfDNA) for evaluating peritoneal effluent in PD patients with and without peritonitis. Study Design Prospective cohort study Setting & Participants We prospectively characterized cell-free DNA (cfDNA) in 68 peritoneal effluent samples obtained from 33 patients receiving peritoneal dialysis at a single center study from September 2016 to July 2018. Outcomes Peritoneal effluent cfDNA characteristics, microbial and human cfDNA characteristics in culture-confirmed peritonitis and culture-negative peritonitis. Analytical Approach Descriptive statistics, detection of microbial cfDNA in culture-confirmed peritonitis and culture-negative peritonitis. Results Metagenomic sequencing of cfDNA was able to detect and identify bacterial, viral, and eukaryotic pathogens in the peritoneal effluent from PD patients with culture-confirmed peritonitis as well as patients with recent antibiotic usage and in cases of culture-negative peritonitis. Limitations Parallel cultures not obtained in all peritoneal effluent specimens. Conclusions Metagenomic cfDNA sequencing of peritoneal effluent can identify pathogens in PD patients with peritonitis, including culture-negative peritonitis.