Purpose <p>Knee osteoarthritis (OA) is a major cause of chronic disability worldwide, affecting millions and posing a substantial public health burden. It leads to progressive cartilage degeneration, inflammation, and impaired joint function, underscoring the need for early diagnostic biomarkers and insight into contributory factors such as parasitic infections. Recent evidence suggests a connection between knee OA and low-grade intestinal inflammation, as well as alterations in the gut microbiota. We aimed to examine synovial fluid for parasitosis (SP) among osteoarthritic patients (OPs) and to detect associated biomarkers.</p> Methods <p>Synovial fluid samples were aspirated and divided into three portions. The first portion was spread to make thin films and stained with various stains. The second portion was centrifuged, and the resulting pellets were inoculated using Jones’ media. The third one was analyzed using the enzyme-linked immunosorbent assay (ELISA) technique to assess Tumor necrosis factor (TNF-α) and matrix metalloproteases-9 (MMP9) biomarkers.</p> Results <p>Synovial parasitosis was detected in 57% of OA patients, with Blastocystis sp. being the most prevalent (40%), followed by microsporidia (16%). Corticosteroid-treated (CST) patients had a higher prevalence of SP, while TNF-α and MMP9 levels were elevated in infected compared with noninfected patients. Elevated biomarker levels correlated with infection multiplicity, indicating inflammatory activation.</p> Conclusions <p>This study highlights a significant prevalence of SP among OPs (57%), with Blastocystis sp. being the most common parasite. CST-treated patients had a higher SP than those with non-CST. Elevated TNF-α and MMP-9 levels could be biomarkers for identifying patients at risk for disease progression due to SP. Further research should be conducted using molecular techniques, along with studies utilizing antiparasitic treatment among infected OA patients.</p>