The antibacterial properties of modified silk fibroin microfibers (SF MFs) have been widely studied. Among various modifications, integration of silver nanoparticles (Ag NPs) and SF MFs has garnered significant attention due to the broad‐spectrum antibacterial activities and long‐term antibacterial effect of Ag nanomaterials. However, the traditional introduction of reducing agents or other additives during the synthesis of Ag‐SF composite MFs potentially affects their structure and antibacterial properties. Facile, green and effective methods for the preparation of Ag‐SF MFs with enhanced antibacterial properties are therefore highly desired. In this study, Ag NPs were uniformly in‐situ deposited onto the optimized SF MFs by adjusting the pH and duration conditions under the guidance of green chemistry. The loaded Ag NPs have a good dispersibility and an average size of ~10 nm. The stability of SF MFs after the deposition of Ag NPs and the crystalline features of the loaded Ag NPs have been carefully investigated. Moreover, antibacterial experiments confirmed that Ag‐SF MFs exhibited superior antibacterial activities. After co‐incubating Ag‐SF MFs with L929 cells, the cell viability reached 90%, demonstrating the great biocompatibility of the modified fibers. This green in‐situ synthetic method will promote the further medical use of Ag‐SF MFs in antibacterial fields.