This study investigated the fabrication, characterization, hemostatic efficacy, biocompatibility, and bioactivity of chitosan-based hemostatic agents. The effects of pH, mixing ratio, and sonication on polyelectrolyte complexation of chitosan with casein and gelatin were studied to form nanofibers through self-assembly. Surface charge and fiber diameter were also investigated for improving the hemostatic efficacy through platelet adhesion and rapid water absorption. The biocompatibility and bioactivity of chitosan-based hemostatic agent studied in rat model. The results showed that increasing the chitosan percentage in the formulation could increase the fiber diameter (r = 0.90) in the PEC complex. Decreasing the fiber diameter increased (r= 0.97) hemostatic efficacy at in vitro conditions. The chitosan and gelatin PEC nanofibers could increase the mechanical strength of PEC-induced blood clots {(68.6 ± 6.4 kPa (CG30) vs. 14 ± 1.2 kPa (Celox&trade)}. The chitosan-based PECs showed bacteriostatic properties against Gram +ve S. aureus and Gram &ndashve E. coli but acquired the bactericidal activity upon incorporation of silver or zinc oxide nanoparticles. Zinc oxide nanoparticle incorporated nanofibrous PEC also showed excellent bioactivity by promoting enzymes such as alkaline phosphatase, lactate dehydrogenase, malate dehydrogenase, and glutamate dehydrogenase activity in rat skin. The optimized chitosan-based hemostat could clot the goat blood within 10 seconds at in vitro conditions by promoting platelet aggregation and contact activation pathways. This also could stop bleeding within 10 seconds to form a stable clot in rat femoral artery model in vivo. In conclusion, this present study reports optimization and characterization of chitosan-based bioactive hemostatic agents with anti-microbial properties that could clot blood within 10s under both in vitro and in vivo conditions and were biocompatible and safe for use.