Cytomegalovirus (CMV)-associated colitis reflects the adverse impact of CMV reactivation on ulcerative colitis (UC). Its diagnosis requires the detection of viral markers in intestinal biopsies sampled during endoscopy, which may constitute invasive and expensive analyses. Moreover, less than 30% of acute flare-ups in steroid refractory UC are associated with CMV colitis. This retrospective study aimed to identify non-invasive factors that are predictive of CMV reactivation, and was conducted from 2014 to 2019 in a cohort of UC patients consulting at the University Hospital of Saint-Etienne, France. Patient characteristics, disease activity, immunosuppressive treatment and tissue CMV DNA load were collected at the time of UC relapse. Factors potentially associated with CMV reactivation were analyzed through a multivariate analysis. A total of 173 UC patients providing 323 pairs of intestinal biopsies were analyzed. In the CMV seropositive subgroup, a Mayo endoscopic score ≥2 (OR 2.553, 95% CI 1.353-4.818, p = 0.004) was identified as a predictive factor of CMV colitis in the multivariate analysis; in contrast, biological parameters exhibited no predictive value. In addition, the use of anti-TNFα monoclonal antibodies was associated with a reduced risk of CMV reactivation (OR 0.384, 95% CI 0.158-0.935, p = 0.035). Intestinal biopsies appear to be unavoidable for assessing disease activity and CMV reactivation in UC patients.