Biofabrication of hierarchical tissues with features ranging various size ranges and controllable anisotropy remains a challenge in 3D-bioprinting. To overcome this hurdle, the application of multi-functional microfibers acting as cell-instructive bioink additive, recently gained particular attention. In this work, we investigate a microfluidic spinning process for the fabrication of collagen microfibers with adjustable diameters ranging from 5 to 50 μm. The thread was collected on a rotating winder and fragmented into microfibers of defined length (60-300 μm). By integrating microfiber fragments into an agarose-hyaluronan hydrogel, fine-tuning of its viscosity range (10-1000 mPa∗s), and thus the precise control of the extruded strands' diameter (0.3-1.4 mm) was achieved. While remaining strong shear-thinning behavior (n-value 0.6), E-modulus and yield stress were decreased in fiber-filled hydrogel, hinting at an interaction of agarose polymer chains with microfibers. Remarkably, the orientation of collagen microfibers could be directed either parallel or orthogonal to the printing path. This allows the biofabrication of hydrogel structures with adjustable domains of defined anisotropy. Finally, the fibers showed excellent biofunctionality both in 2D and 3D. Besides a high degree of alignment of individual cells along the microfiber axis (>80 % of cells), hMSCs built a dense, branched network in 3D. Moreover, PC12 and C2C12 were successfully differentiated in 2D and 3D. Specifically, neurite length was higher on smaller fiber diameters, even spanning non-adjacent clusters. Elongated, multi-nuclei myotubes were formed, indicating C2C12 differentiation. In summary, the work demonstrates the great potential of 3D-bioprinting in cross-scale organization of fragmented collagen microfibers.