The use of constructed wetlands as a valuable and attractive method for combined sewer overflow (CSO) treatment has been demonstrated in several studies. In Portugal, a Mediterranean country having usually a long dry period, there are still no applications of this technology. The purpose of this research is to gather information and know-how required for the design and management of this type of infrastructure. A pilot-scale experimental setup for CSO treatment was installed and evaluated in situ, in terms of organic matter, total suspended solids and microorganism removal with emphasis on the results of the start-up. After 1 day of retention average removal efficiencies of 73–79% and 82–89% were obtained in terms of chemical oxygen demand (COD) and total suspended solids (TSS), respectively. During the remaining retention time a slower removal was observed. After 7 days, the COD removal efficiencies reached 86–91% and the TSS removal efficiencies reached 93–97%. On average, after 1 day, reductions of 1.2–2.0 log and 1.9–2.4 log, respectively, for total coliforms and Enterococcus were observed. For a retention time of 7 days these reductions attained 4.0–4.9 log and 4.4–5.3 log, respectively.