Combination chemotherapy is considered an effective strategy to inhibit tumor growth. When nanocarriers are utilized in the process, both physical and chemical modifications are usually required to enable the co‐loading of chemotherapeutic drugs with different properties. Here, beta‐sheet‐rich silk nanofibers were co‐loaded with hydrophilic doxorubicin (DOX) and hydrophobic paclitaxel (PTX) through a sequential physical blending‐centrifugation‐blending process. The ratio and amount of DOX and PTX on the nanofibers were regulated independently to optimize cooperative interaction by changing the amount of drug added. Both PTX and DOX were immobilized on the same nanofibers to avoid burst release problems. Besides the water‐insoluble PTX, more than half of the DOX remained fixed on the nanofibers for more than 28 days, which facilitated the co‐internalization of both DOX and PTX by tumor cells in vitro. Changing the ratio of co‐loaded DOX and PTX achieved optimal combination therapy in vitro. The DOX‐PTX co‐loaded nanofibers were also assembled into injectable hydrogels to facilitate in situ injection around tumor tissues in vivo. Effective long‐term inhibition was achieved for tumors treated with DOX‐PTX co‐loaded hydrogels, superior to those treated with both free DOX and PTX, and hydrogels loaded with only DOX or PTX. Considering the mild and controllable physical loading process and superior loading capacity for both hydrophilic and hydrophobic ingredients, these injectable silk nanofiber hydrogels are promising carriers to deliver multiple drug types simultaneously in situ, enhancing combination chemotherapies towards clinical applications.This article is protected by copyright. All rights reserved