We report the role of relative lymphocyte count (RLC) as a potential biomarker with prognostic impact for catumaxomab efficacy and overall survival (OS) based on a post hoc analysis of the pivotal phase II/III study of intraperitoneal catumaxomab treatment of malignant ascites. The impact of treatment and RLC on OS was evaluated using multivariate Cox models. Kaplan-Meier and log-rank tests were used for group comparisons. Survival analyses were performed on the safety population patients with paracentesis plus ≥ 1 dose of catumaxomab (n = 157) and paracentesis alone (n = 88). Determination of the optimal cutoff value for RLC was based on five optimality criteria. OS was significantly longer with catumaxomab versus paracentesis alone (P = 0.0219). The 6-month OS rate with catumaxomab was 28.9% versus 6.7% with paracentesis alone. RLC had a positive impact on OS and was an independent prognostic factor (P < 0.0001). In patients with RLC > 13% (n = 159: catumaxomab, 100 and control, 59), catumaxomab was associated with a favorable effect on OS versus paracentesis alone (P = 0.0072), with a median/mean OS benefit of 41/131 days and an increased 6-month survival rate of 37.0% versus 5.2%, respectively. In patients with RLC ≤ 13% at screening (n = 74: catumaxomab, 50 and control, 24), the median (mean) OS difference between the catumaxomab and the control group was 3 (16) days, respectively (P = 0.2561). OS was significantly improved after catumaxomab treatment in patients with malignant ascites. An RLC > 13% at baseline was a significant prognostic biomarker.