Due to the high surface area, porosity, and rigidity, applications of nanofibers and nanosurfaces have developed in recent years. Nanofibers and nanosurfaces are typically produced by electrospinning method. In the production process, determination of average fiber diameter is crucial for quality assessment. Average fiber diameter is determined by manually measuring the diameters of randomly selected fibers on scanning electron microscopy (SEM) images. However, as the number of the images increases, manual fiber diameter determination becomes a tedious and time consuming task as well as being sensitive to human errors. Therefore, an automated fiber diameter measurement system is desired. In the literature, this task is achieved by using image analysis algorithms. Typically, these methods first isolate each fiber in the image and measure the diameter of each isolated fiber. Fiber isolation is an error-prone process. In this study, automated calculation of nanofiber diameter is achieved without fiber isolation using image processing and analysis algorithms. Performance of the proposed method was tested on real data. The effectiveness of the proposed method is shown by comparing automatically and manually measured nanofiber diameter values.