Nanobodies (Nbs) hold great promise as next-generation cancer immunotherapies, but their efficacy is hindered by their poor pharmacokinetics and the inability to trigger Fc-mediated immune killing functions. To address these limitations, we designed and synthesized rhamnolipid-modified Nbs as a type of antibody-recruiting molecule by site-specifically conjugating EGFR-targeting Nb 7D12 to a series of rhamnolipid derivatives, and their biological profiles were evaluated in vitro and in vivo. Investigation of the structure-activity relationship revealed that the number of rhamnose (Rha) units and the length of the PEG linker in the conjugates affected anti-tumor activities. Conjugate R5, which contained two Rha units and a PEG