Aerosol Jet® printing (AJ®P) is a direct writing printing technology which deposits functional aerosolized solutions on free-form substrates. Its potential has been widely adopted for two-dimensional (2D) microscale constructs in printed electronics (PE), and it is rapidly growing toward surface structuring and biological interfaces. However, limited research has been devoted to its exploitation as a three-dimensional (3D) printing technique. This paper investigates 3D AJ®P capabilities of three inks along with a comparison of their abilities and limitations by employing three AJ®P 3D strategies (continuous jet deposition, layer-by-layer, point-wise). In particular, 3D microstructures of increasing complexity based on a silver nanoparticle (AgNPs)-, a poly(3,4-ethylenedioxythiophene)polystyrene sulfonate (PEDOT:PSS)-, and a collagen-based ink are here investigated at various aspect ratios and resolutions. Results show the possibility to print not only arrays of micro-pillars of different aspect ratios (AgNPs-ARs ∼ 20, PEDOT:PSS-ARs ∼ 7, collagen-ARs ∼ 3), but also dense and complex (but low reproducible) leaf- or flake-like structures (especially in AgNPs), and lattice units (collagen). This study demonstrates that the fabrication of 3D AJ® printed microstructures firmly depend on the printing parameters and the ink (co–)solvents fast-drying phenomena during printing. Moreover, it provides guidelines about ink development and print strategies for 3D AJ®P micro-structuring, opening its adoption in a vast range of applications in life science (tissue engineering, bioelectronic interfaces), electronics, and micromanufacturing.