Waterproof textiles have wide applications in many fields such as sportswear, protective clothing, and orthopedic dressing. Breathability is an important factor of clothing comfort. Nanofibrous layers have application in these fields due to their interconnected porous area and high surface area. In this work, two different nanofiber layers were fabricated and examined as waterproof and breathable layers. Polyurethane (PU) and PU/nylon 66 (hybrid) nanofiber layers were produced via electrospinning set up with different electrospinning durations. Some of the samples’ characteristics such as tensile strength, microindentation, air permeability, water vapor permeability (WVP), and contact angle of water were investigated. Moreover, a novel approach was applied for determining the performance of layers against acidic water to simulate acidic rain. The results show that the tensile strength, indentation force, and acidic waterproof ability of the layers increased with increasing the process duration, that is, while air permeability was decreased simultaneously. This work shows that the required force for indentation and strength of the hybrid layer was less than that of PU nanofiber layer. Also, the electrospun hybrid layers show better air permeability than the PU membrane but still have lower WVP, which affects the breathability of the layer.