Medically approved sterility methods should be a major concern when developing a polymeric scaffold, mainly when commercialization is envisaged. In the present work, poly(lactic acid) (PLA) fiber membranes were processed by electrospinning with random and aligned fiber alignment and sterilized under UV, ethylene oxide (EO), and γ-radiation, the most common ones for clinical applications. It was observed that UV light and γ-radiation do not influence fiber morphology or alignment, while electrospun samples treated with EO lead to fiber orientation loss and morphology changing from cylindrical fibers to ribbon-like structures, accompanied to an increase of polymer crystallinity up to 28%. UV light and γ-radiation sterilization methods showed to be less harmful to polymer morphology, without significant changes in polymer thermal and mechanical properties, but a slight increase of polymer wettability was detected, especially for the samples treated with UV radiation. In vitro results indicate that both UV and γ-radiation treatments of PLA membranes allow the adhesion and proliferation of MG 63 osteoblastic cells in a close interaction with the fiber meshes and with a growth pattern highly sensitive to the underlying random or aligned fiber orientation. These results are suggestive of the potential of both γ-radiation sterilized PLA membranes for clinical applications in regenerative medicine, especially those where customized membrane morphology and fiber alignment is an important issue.