A recent study has suggested that irrigation with povidone–iodine solution after knee arthroplasty significantly decreases rates of post-operative surgical site infection. However, there is only limited knowledge of potential chondrotoxic effects on the residual cartilage in patients with partial knee arthroplasties or unresurfaced patella in total knee arthroplasty. Macroscopically normal bovine cartilage explants (n = 42) were exposed to different povidone–iodine concentrations for 1, 3 or 6 min, as well as saline control. The viability of superficial chondrocytes was measured by a Live/Dead cytotoxicity assay. Chondrotoxicity correlated positively with the length of exposure, regardless of the concentration. The extent of superficial chondrocyte death was significantly greater at higher concentrations of povidone–iodine solutions. 0.35% povidone–iodine solution was the least chondrotoxic of all concentrations, but still reduced cell viability significantly if applied for longer than 1 min. Our data suggest that povidone–iodine solution at all tested concentrations has a pronounced chondrotoxic effect on the superficial cartilage layer when used for time periods longer than 1 min.