WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: Warts, Hypogammaglobulinemia, Infections and Myelokathexis. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4. Consequently, these CXCR4-WHIM mutants have a gain of function in response to its ligand CXCL12, which results in abnormal leukocyte migration and thereby immune system dysfunctions. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific Nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies(®) are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific Nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small molecule clinical candidate AMD3100. The CXCR4-targeting Nanobodies displace CXCL12 binding and bind CXCR4-WT and -WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The Nanobodies' epitope was mapped to the extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent Nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of HPV-immortalized cell lines in response to CXCL12 was effectively inhibited by the Nanobodies. Based on these in vitro results, we conclude that CXCR4 Nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases like WHIM syndrome.