ABSTRACT 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. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43657.