Purpose: Soft tissue, bone and joint infections are severe complications in orthopedic and traumatological surgery. Lavanox (0.08% NaOCl) and Irrisept (0.05% chlorhexidine gluconate, CHG) are industrially produced antiseptic solutions commonly used in infection treatment. Regarding this clinical indication, the microbicidal effect is often investigated, but toxicity to osteoblasts has rarely been examined. This is important to decide whether these solutions should be used in septic situations in which bone healing must take place. The hypothesis of the present study is that NaOCl and CHG are cytotoxic to osteoblasts even after a short exposure time. Methods: Human osteoblasts were isolated from donors with osteoarthritis during total knee and hip arthroplasty. Cells were cultivated and treated with both antiseptic solutions for 2, 5 and 10 min in different dilutions. Toxicity was quantified by counting cells, lactate dehydrogenase (LDH) expression, spectrophotometric quantification via XTT assay and FDA/PI fluorescence microscopy. Results: Analyzing viable cells after treatment with both antiseptics showed a significant decrease in viable cells through LDH expression test, XTT assay, fluorescence microscopy and light microscopy, depending on concentration. The time dependence showed a trend to more cell death at longer exposure times, without significance. Conclusion: Toxic effects on osteoblasts were shown after treatment with 0.08% NaOCl and 0.05% CHG after an exposure time of 2 min which also was concentration dependent. There was no difference in cytotoxicity between both antiseptics. In conclusion, these antiseptic solutions may be used with caution in situations requiring bone healing. Trial registration number Local ethics committee registration number: 5176–07/16