We produced an anatomically and dielectrically realistic phantom of the axillary region to enable the experimental assessment of Axillary Lymph Node (ALN) imaging using microwave imaging technology. We segmented a thoracic Computed Tomography (CT) scan and created a computer-aided designed file containing the anatomical configuration of the axillary region. The phantom comprises five 3D-printed parts representing the main tissues of interest of the axillary region for the purpose of microwave imaging: fat, muscle, bone, ALNs, and lung. The phantom allows the experimental assessment of multiple anatomical configurations, by including ALNs of different size, shape, and number in several locations. Except for the bone mimicking organ, which is made of solid conductive polymer, we 3D-printed cavities to represent the fat, muscle, ALN, and lung and filled them with appropriate tissue-mimicking liquids. Existing studies about complex permittivity of ALNs have reported limitations. To address these, we measured the complex permittivity of both human and animal lymph nodes using the standard open-ended coaxial-probe technique, over the 0.5 GHz–8.5 GHz frequency band, thus extending current knowledge on dielectric properties of ALNs. Lastly, we numerically evaluated the effect of the polymer which constitutes the cavities of the phantom and compared it to the realistic axillary region. The results showed a maximum difference of 7 dB at 4 GHz in the electric field magnitude coupled to the tissues and a maximum of 10 dB difference in the ALN response. Our results showed that the phantom is a good representation of the axillary region and a viable tool for pre-clinical assessment of microwave imaging technology.