Debilitating symptoms of recurrent Clostridioides difficile infection (rCDI) often lead to long-term effects on health-related quality-of-life (HRQOL). In ECOSPOR III, SER-109, an investigational oral microbiome therapeutic, was superior to placebo in reducing rCDI. We investigated the validity, reliability, and responsiveness of a 32-item, CDI-specific questionnaire-the Clostridium difficile Quality of Life Survey (Cdiff32)-across mental, physical, and social domains in patients with rCDI. In this post hoc analysis of a phase 3 clinical trial, 182 outpatients with rCDI completed Cdiff32 and EQ-5D at baseline and at 1 and 8 weeks. Cdiff32 was evaluated for item performance, internal reliability, and convergent validity. To assess known-groups validity, Cdiff32 scores were compared by disease recurrence status at week 1; internal responsiveness was evaluated in the nonrecurrent disease group by 8 weeks by means of paired t test. All 182 patients (mean age [standard deviation], 65.5 [16.5] years; 59.9% female) completed baseline Cdiff32. Confirmatory factor analysis identified 3 domains (physical, mental, and social relationships) with good item fit. High internal reliability was demonstrated (Cronbach α = 0.94 with all subscales >0.80). Convergent validity was evidenced by significant correlations between Cdiff32 subscales and EQ-5D (r = 0.29-0.37; P < .001). Cdiff32 differentiated patients by disease recurrence status at week 1 (effect sizes, 0.38-0.42; P < .05 overall), with significant improvement from baseline through week 8 in patients with nonrecurrent disease at week 1 (effect sizes, 0.75-1.02; P < .001 overall). Cdiff32 is a valid, reliable, and responsive disease-specific HRQOL questionnaire that is fit for purpose for interventional treatment trials. The significant improvement in patients with nonrecurrent disease by 8 weeks demonstrates the negative impact of rCDI on HRQOL.