The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity by flow cytometry, western blotting and immunohistochemistry. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, E134Bf. Kinetic analysis of the interactions of E134Bf with the canine osteosarcoma cell line (D-17) and canine fibroblastic cell line (A-72) cells was conducted by flow cytometry. The K_D for the interaction of E134Bf with the D-17 and A-72 cells was 5.5 × 10⁻¹⁰ M and 6.0 × 10⁻¹⁰ M, respectively, indicating that E134Bf exhibits high affinity for D-17 and A-72 cells. Furthermore, E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 and A-72 cells. In vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.