The objective of this study was to determine if vaginal stiffness index, an in vivo vaginal biomechanical property, is correlated with pelvic floor disorder symptom distress, impact on quality of life, or sexual function as measured by disease-specific quality-of-life scales. Forty-eight women completed validated quality-of-life scales (pelvic floor distress inventory-short form, pelvic floor impact questionnaire, and pelvic organ prolapse/urinary incontinence sexual questionnaire) and underwent in vivo vaginal biomechanical testing. After bivariate relationships between vaginal stiffness index and demographic, obstetric, and gynecologic variables were explored, multiple linear regression controlling for pelvic organ prolapse quantitative (POP-Q) stage of prolapse was performed. The vaginal stiffness index was inversely correlated with pelvic organ prolapse distress severity (POPDI-6) after controlling for POP-Q stage of prolapse (p = 0.011, r = 0.67, r (2) = 0.450, beta = -2.3). These findings provide initial evidence for the construct validity of in vivo vaginal biomechanical testing for pelvic organ prolapse evaluation because an increasing vaginal stiffness index is correlated with decreasing symptomatic and anatomic severity of disease.