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Scaffolds play a crucial role in bone tissue engineering to support the defect area through bone regeneration and defect reconstruction. Promising tissue regeneration without negative repercussions and avoidance of the lifelong presence inside the body make bioresorbable metals prosper in the field of regenerative medicine. Recently, Zn and its alloys have emerged as promising biodegradable materials for their moderate degradation rate and satisfactory biocompatibility. Nevertheless, it is very challenging for cells to adhere and grow over the Zn surface alone, which influences the tissue-implant integration. In this study, an attempt has been made to systematically investigate the bioactivity responses in terms of in vitro hemocompatibility, cytotoxicity, antibacterial activity, and in vivo biocompatibility of newly developed Zn–2Cu–0.5Mn/Mg alloy scaffolds with different surface roughness. The rough surface of Zn–2Cu–0.5Mg shows the highest degradation rate of 0.16 mm/yr. The rough surface exhibits a prominent role in the adsorption of protein, further enhancing cell adhesion. Concentration-dependent alloy extract shows the highest cell proliferation for 12.5% of the extract with a maximum cell viability of 101% in Zn–2Cu–0.5Mn and 108% in Zn–2Cu–0.5Mg after 3 d. Acceptable hemolysis percentages (less than 5%) with promising anticoagulation properties are observed for all of the conditions. Enhanced antibacterial (Staphylococcus aureusand Escherichia coli) activity due to a significant effect of ions illustrates the maximum killing effect on the bacterial colony for the rough Zn–2Cu–0.5Mg alloy. In addition, it is observed that for rough Zn–2Cu–0.5Mn/Mg alloys, the inflammatory response is minimal after subcutaneous implantation, and neo-bone tissue forms in the defect areas of the rat femur with satisfactory biosafety response. The osseointegration property of the Zn–2Cu–0.5Mg alloy is comparable to that of the Zn–2Cu–0.5Mn alloy. Therefore, the rough surface of the Zn–2Cu–0.5Mg alloy has the potential to enhance biocompatibility and promote better osseointegration activity with host tissues for various biomedical applications.