The fabrication of biomedical composite materials with macroporous structures and unique topographical cues has been widely investigated to achieve successful bone regeneration. In this study, porous biocomposites consisting of microfibrous bundles fabricated using an electrohydrodynamic direct printing process were prepared. The fibrous composite structure was composed of a fibrous matrix structure of polycaprolactone/a-tricalcium phosphate and collagen coated in fibrous biocomposites. Various cellular activities, cell proliferation, and osteogenic differentiation in biocomposites have been investigated using preosteoblasts (MC3T3-E1). The in vitro results demonstrated that biocomposites with the microfibrous topographical cue significantly improved various cellular responses, including cell proliferation and mRNA expression levels of osteoblastic genes of MC3T3-E1 cells, compared to biocomposites without a fibrous topography surface that were fabricated through normal 3D printing. This phenomenon could be attributed to the fibrous structure of composites that stimulated cultured cells, thereby activating extracellular signal-related kinases and p38 signaling pathways. To observe the ability of biocomposites for bone regeneration, a rat calvarial defect model was used; the fibrous biocomposite showed significantly higher level of new bone formation in comparison with the 3D-printed control, a biocomposite without fibrous topographical cues.