Recently, an effective hemostatic dressing requirement has become a major problem in both the military and civilian world. Available hemostatic agents are too expensive, ineffective, unsafe, or complicated to use. Here, we evaluated the hemostatic efficacy of a nano/micro bilayer hemostatic dressing including a porous sublayer from chitosan (CTS) and bacterial cellulose (BC) and a nanofibrillar upper layer from silk fibroin (SF). In addition, several active agents rolled in coagulation cascade [vitamin K (Vit K), protamine sulfate (PS), kaolin (Kao), etc.] were doped to the sublayer of bilayer hemostatic agent and their activities were compared via in vivo and in vitro tests. Lactate dehydrogenase (LDH) activity test results demonstrated that BC/CTS, SF-coated BC/CTS, and Vit K K/BC/CTS, SF/phosphatidylcholine (PC)-coated BC/CTS showed higher LDH activity compared to standard gauze (p<0.005). In a femoral artery bleeding rat model, SF-coated PS/BC/CTS significantly reduced bleeding time (80 +/- 0.3 s) compared to standard gauze and kaolin-doped group (p<0.005). Blood loss and mortality rate with 2.3 g and 37.5% SF-coated BC/CTS and SF-coated PS/BC/CTS showed increased efficacy in achieving hemostasis compared to standard gauze. All the prepared hemostatic dressings except for kaolin-doped BC/CTS and standard gauze induced no inflammatory reaction in the tissue. All these data suggest that active agent-doped BC/CTS/SF-based bilayer hemostatic dressings have a great influence on the resulting hemostatic action. (c) 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43657.