Optical microscopy has been one of the most important tools for visualizing biological samples since the seventeenth century. Recently, with the advances in femtosecond laser technology, all the nonlinear optical processes have now been included as optical microscopy methods, and second harmonic generation (SHG) microscopy has emerged as a powerful new optical imaging tool with applications in medicine and biology. Here we use SHG microscopy to obtain images of 76 prostate biopsies on histological slides. Multiple samples from the excised prostates of patients who underwent a radical prostatectomy were evaluated. The samples were collected from prostate positions as in needle biopsy procedures. The results show the collagen fiber architecture among malignant acini, and analysis of the fiber orientation in the images reveals that the collagen fibers become more aligned at higher malignancy grades. Furthermore, we find that the degree of fiber alignment correlates directly with the Gleason patterns.