The biocompatibility and osteogenic potential of four fibrous scaffolds prepared by electrospinning of poly(ε-caprolactone) (PCL) was studied with MG-63 osteoblast cells. Two different kinds of scaffolds were obtained by adjustment of spinning conditions, which were characterized as nano- or microfibrous. In addition of one nanofibrous, scaffold was made more hydrophilic by blending PCL with Pluronics F 68. Scaffolds were characterized by scanning electron microscopy and water contact angle measurements. Morphology and growth of MG63 cells seeded on the different scaffolds were investigated by confocal laser scanning microscopy after vital staining with fluorescein diacetate and by colorimetric assays. It was found that scaffolds composed of microfibres stipulated better growth conditions for osteoblasts probably by providing a real three-dimensional culture substratum, while nanofibre scaffolds restricted cell growth predominantly to surface regions. Osteogenic activity of cells was determined by alkaline phosphatase (ALP) and o-cresolphthalein complexone assay. It was observed that osteogenic activity of cells cultured in microfibre scaffolds was significantly higher than in nanofibre scaffolds regarding ALP activity. Overall, one can conclude that nanofibre scaffold provides better conditions for initial attachment of cells but does not provide advantages in terms of scaffold colonization and support of osteogenic activity compared to scaffolds prepared from microfibres.