Interfaces between layers in 3D-printed elements produced by extrusion-based material deposition were investigated on both macro- and micro-scales. On the macro-scale, compression and flexural tests were performed on two 3D-printable cement-based compositions (3PCs), namely Mixtures C1 (with Portland cement as sole binder) and C2 (containing pozzolanic additives) at testing ages of 1 day and 28 days. The influences of binder composition and time interval between layers on layer-interface strength were critically analyzed. The investigated time intervals were 2 min, 10 min and 1 day. The investigations revealed that Mixture C2 exhibited lower degrees of anisotropy and heterogeneity as well as superior mechanical performance in comparison to Mixture 1. In particular, Mixture C2 showed a less pronounced (below 25%) decrease in interface bond strength as observed in flexural tests for all time intervals under investigation. In contrast, the decrease in flexural strength measured for C1 specimens amounted to over 90% due to the higher porosity at the interfaces of the printed concrete layers. Microscopic observations supported the findings of the macroscopic investigations. SEM images also delivered additional information on morphology of interfacial defects as well as “self-healing”.