Chronic kidney disease (CKD) affects 10% of the world’s population. Uremic toxins, such as indoxyl sulfate (IS), p-Cresylsulfate (PCS) and indole acetic acid (IAA), are not sufficiently removed by conventional hemodialysis (HD) and have been associated with inflammation, poor quality of life, bone mineral disease (BMD) and endothelial injury. Online hemodiafiltration (OL-HDF) may promote greater clearance of uremic toxins than HD. However, there are few studies evaluating the effect of OL-HDF on serum levels of IS, PCS, IAA, and biomarkers associated with inflammatory, endothelial, and bone and mineral disorder in the elderly population. We evaluated the effect of 6 months of OL-HDF on the serum concentration of uremic toxins, biomarkers of inflammation, endothelial and bone mineral disorder in older patients on OL-HDF. IS, PCS, and IAA were measured by high-performance liquid chromatography. We included 31 patients (77.4 ± 7.1 years, 64.5% male, 35.5% diabetic, on maintenance dialysis for 45 ± 20 days). From baseline to 6 months there was a decrease in serum concentration of IS but not PCS and IAA. We found no change in serum concentration of inflammatory, endothelial, or mineral and bone biomarkers. In summary, OL-HDF was capable to reduce IS in older patients. Whether this reduction may have an impact on clinical outcomes deserves further evaluation.