Arroyo Ayuí Grande is a stream in the Concordia department of Entre Ríos Province, Argentina. It originates at an elevation of approximately 42 meters in the foothills of the Cuchilla Grande, a few kilometers northeast of Los Charrúas, and flows generally southeast for 26.38 kilometers before emptying into the Río Uruguay roughly 1,800 meters downstream of the Salto Grande dam, within the municipal limits of Concordia. Including its source tributaries, the total watercourse measures approximately 34.67 kilometers. The stream forms an island at one point along its course. Its principal tributary is the Arroyo Loma de Negra (15.09 km), followed by the Arroyo Seco (8.43 km), along with four additional smaller tributaries.

The arroyo flows through gallery forest that forms part of the broader Selva Misionera, also known as the Paranaense forest. Riparian vegetation along the banks includes seibo, canelón, palma pindó, laurel, and curupí. A private natural reserve of 215 hectares was established in July 2000 in the area where Ruta Nacional N° 14 crosses the arroyo, with the aim of protecting this gallery forest corridor. A conservation corridor initiative along the full length of the stream involves landowner agreements that prohibit trammel net fishing, cutting of native timber, application of agrochemicals within 50 meters of the water, hunting, obstruction of the watercourse, and washing of machinery in the stream. Paddlers should be aware of these conservation restrictions when planning trips.

Fish species found in the arroyo include Golden dorado, Boga, and Trahira, reflecting the stream's connection to the broader Río Uruguay watershed. The mouth of the arroyo at the Río Uruguay is located at approximately 31°17′S, 57°57′W, near the locality known as La Tortuga Alegre.