The potential use as a bone substitute material of a three-dimensional bioactive glass fiber scaffold composed of Na(2)O-K(2)O-MgO-CaO-B(2)O(3)-P(2)O(5)-SiO(2) (BG1) was investigated in this work. Scaffolds were pre-treated with simulated body fluid (SBF) to promote the formation of two different bone-like apatite layers on their surfaces. The topography and roughness of the deposited layers were assessed by scanning electron microscopy (SEM), while the chemical composition and structure using X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy, respectively. Based on surface analysis, the bioactive glass surfaces were ranked from smoothest to roughest: 0 SBF (untreated), 1x SBF and 2x SBF. A calcium-deficient carbonated hydroxyapatite (HCA) layer was present on both SBF-treated scaffolds, with higher number and larger bone-like apatite nodule formation in the 2x SBF case. MC3T3-E1 preosteoblasts showed a more flattened morphology and higher cell proliferation on the nontreated scaffolds; whereas, cells were more elongated and had higher osteoblastic activity on SBF-treated samples. In vivo results in a rabbit calvarial bone defect model showed enhanced bone formation with SBF pretreated scaffolds, compared with untreated ones, commercially available Perioglass particles and empty defects. Our findings demonstrate that the formation of a rough HCA layer on bioactive glass porous scaffolds enhanced preosteoblast maturation in vitro, as well as bone formation in vivo.