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This prospective pilot study examined the association between microorganisms and knee osteoarthritis by identifying pathogens in the synovial membrane, synovial fluid, and blood samples from two patients with primary bilateral knee osteoarthritis, using metagenomic next-generation sequencing (mNGS). Intraoperatively, during routine knee arthroplasty procedures, we collected the following 12 samples from each patient: two synovial membrane samples, two synovial fluid samples, and two venous blood samples. After DNA isolation and library construction, each sample was subjected to deep whole-genome sequencing using the DNBSEQT17 platform with the read length PE150 as the default. Metagenomic sequencing data were mapped to the NCBI NT database to determine species abundance. The predominant species in all samples tested were classified under the Enterobacterales order, the most abundant being Yersinia enterocolitica. The second and third most common microorganisms detected were Escherichia coli and autotrophic, Gram-negative bacteria Synechococcus sp., which is a bioaerosol component, indicating a risk of inhalation of the toxic metabolites of this latter microorganism. This article provides an initial exploration of mNGS use to study the etiopathogenetic mechanisms of knee osteoarthritis (OA). While our analysis identified bacterial DNA, particularly from Yersinia, further cross-sectional studies in larger populations with and without OA are needed to determine the role of these agents in OA pathogenesis. [ABSTRACT FROM AUTHOR]