Barrier property of materials to microbes is an important aspect in many applications like wound dressings and skin substitutes. Lack of a standard method for the evaluation of microbial barrier property limits the consideration of this important aspect by many bioengineers and biomaterial scientists. Thus, in this study we are intended to develop a standard method for the quantitative assessment of Microbial Barrier Property (MBP) and express this in terms of percentage in comparison with a control which resembles a bare wound or a standard existing wound coverage material. In this study, we have evaluated the microbial barrier property of electrospun materials with different pore spaces and our study showed that this method can be successfully used for the assessment of bacterial barrier property of materials intended for wound dressing and skin substitute applications. Further, the results showed that the MBP will vary with the test conditions. This methodology can be used to measure the microbial barrier property of wide range of materials like skin substitute materials, wound dressings, medical textiles and many other materials where microbial prevention is important. This methodology can also be extended to the specific determination of bacterial, fungal or viral barrier property with minor modifications.