Mesothelin (MSLN) is a glycosyl phosphatidyl inositol-anchored glycoprotein involved in the carcinogenesis and metastasis of some cancers such as ovarian cancer, pancreatic cancer, and mesothelioma. The interaction between cancer antigen 125 (CA125) and MSLN enhances tumor metastasis. MSLNs are highly expressed in cancer and are therefore promising targets for cancer therapy. Variable domains of heavy chain of heavy chain (VHH; also known as nanobodies) monoclonal antibodies (mAbs) from alpacas exhibit unique properties such as high affinity, low immunogenicity, and thermal stability. Herein, we aimed to investigate the effects of anti-MSLN VHH mAbs on MSLN binding affinity. Two anti-MSLN VHH mAb clones (MT1A1 and MT3C2), targeting the extracellular domain of human MSLN, were used. MT1A1 and MT3C2 are specific to human and mouse MSLN. MT1A1 and MT3C2 bound to MSLN are expressed on ovarian, pancreatic, and mesothelioma cancer cells. These antibodies recognize the region (296–390) at the N-terminal of MSLN on the cell surface and relying on their conformation-dependent recognition. MT1A1 and MT3C2 significantly inhibited the MSLN-CA125 interaction. Overall, our results suggest that MT1A1 and MT3C2 are promising anticancer agents.