Poly(l-lactide) electrospun mats with random and aligned fiber orientation and films have been produced with degrees of crystallinity ranging from 0 up to nearly 50%. The overall surface roughness is practically constant irrespective of the sampling areas (1 × 1 µm to 20 × 20 µm) for degrees of crystallinity below 30%, increasing for higher degrees of crystallinity for the larger sampling areas. Further, due to fiber confinement, surface roughness variations are smaller in electrospun mats. Samples with 50% of crystallinity show the lowest osteoblast and the highest fibroblast proliferation. Therefore, it is verified that higher roughness promotes lower osteoblast but higher fibroblast proliferation. The overall results indicate the relevant role of the sub-microenvironment variations associated to the microscale roughness in determining the different cell responses. ; Portuguese Foundation for Science and Technology (FCT); contract grant numbers: C/FIS/UI607/2011, PTDC/CTM-NAN/112574/2009, contract grant numbers: SFRH/BPD/63148/2009, SFRH/BPD/90870/2012, Matepro—Optimizing Materials and Processes, Programa Operacional Regional do Norte, Quadro de Referência Estratégico Nacional” (QREN), “Fundo Europeu de Desenvolvimento Regional” (FEDER); contract grant number: NORTE-07–0124-FEDER-000037.