Calcium sulfate, an injectable and biodegradable bone‐void filler, is widely used in orthopedic surgery. Based on clinical experience, bone‐defect substitutes can also serve as vehicles for the delivery of drugs, for example, antibiotics, to prevent or to treat infections such as osteomyelitis. However, antibiotic additions change the characteristics of calcium sulfate cement. Moreover, high‐dose antibiotics may also be toxic to bony tissues. Accordingly, cefazolin at varying weight ratios was added to calcium sulfate samples and characterized in vitro. The results revealed that cefazolin changed the hydration reaction and prolonged the initial setting times of calcium sulfate bone cement. For the crystalline structure identification, X‐ray diffractometer revealed that cefazolin additive resulted in the decrease of peak intensity corresponding to calcium sulfate dihydrate which implying incomplete phase conversion of calcium sulfate hemihydrate. In addition, scanning electron microscope inspection exhibited cefazolin changed the morphology and size of the crystals greatly. A relatively higher amount of cefazolin additive caused a faster degradation and a lower compressive strength of calcium sulfate compared with those of uploaded samples. Furthermore, the extract of cefazolin‐impregnated calcium sulfate impaired cell viability, and caused the death of osteoblast‐like cells. The results of this study revealed that the cefazolin additives prolonged setting time, impaired mechanical strength, accelerated degradation, and caused cytotoxicity of the calcium sulfate bone‐void filler. The aforementioned concerns should be considered during intra‐operative applications.