Aims. In orthopaedic and trauma surgery, implant-associated infections are increasingly treated with local application of antibiotics, which allows a high local drug concentration to be reached without eliciting systematic adverse effects. While ceftriaxone is a widely used antibiotic agent that has been shown to be effective against musculoskeletal infections, high local concentrations may harm the surrounding tissue. This study investigates the acute and subacute cytotoxicity of increasing ceftriaxone concentrations as well as their influence on the osteogenic differentiation of human bone progenitor cells. Methods. Human preosteoblasts were cultured in presence of different concentrations of ceftriaxone for up to 28 days and potential cytotoxic effects, cell death, metabolic activity, cell proliferation, and osteogenic differentiation were studied. Results. Ceftriaxone showed a cytotoxic effect on human bone progenitor cells at 24 h and 48 h at concentrations above 15,000 mg/l. With a longer incubation time of ten days, subtoxic effects could be observed at concentrations above 500 mg/l. Gene and protein expression of collagen, as well as mineralization levels of human bone progenitor cells, showed a continuous decrease with increasing ceftriaxone concentrations by days 14 and 28, respectively. Notably, mineralization was negatively affected already at concentrations above 250 mg/l. Conclusion. This study demonstrates a concentration-dependent influence of ceftriaxone on the viability and mineralization potential of primary human bone progenitor cells. While local application of ceftriaxone is highly established in orthopaedic and trauma surgery, a therapeutic threshold of 250 mg/l or lower should diminish the risk of reduced osseointegration of prosthetic implants. Cite this article: Bone Joint Res 2021;10(3):218–225