In septic joint surgery, treatment with antiseptics is a standard procedure. Postinfectious degeneration of cartilage often results in early arthritis that necessitates joint replacement, even in young patients. It is still unclear whether antiseptics have only a toxic effect on chondrocytes or also induce late cell death through apoptosis. We hypothesized that commonly used antiseptics (polyhexanide, hydrogen peroxide, and chlorhexidine) induce different stages of apoptosis on human chondrocytes. Human chondrocytes were isolated and cultured. Polyhexanide, hydrogen peroxide, and chlorhexidine were added to the monolayer cultures. Early and late apoptotic cells were analyzed using flow cytometric detection of Annexin V, active caspases, and 7AAD, and fluorescence microscopy using Annexin V and propidium iodide staining. Flow cytometric analysis revealed an increase of Annexin V and active caspases expression of human chondrocytes after incubation with polyhexanide, hydrogen peroxide, and chlorhexidine. Fluorescence microscopy demonstrated a high number of apoptotic cells. Our data show that polyhexanide and chlorhexidine only promote the early stages of apoptosis without any differences in toxicity. Conversely, hydrogen peroxide induces early and later stages of apoptosis on primary human chondrocytes in vitro.