The second section of the Orto Botanico covers the central portion of the garden and slowly rises in elevation in the direction of the Janiculum Hill. Winding paths, water features, and a variety of planted species compose this middle area. Evident plant orders include palm, pine, and cypress trees, as well as diverse flora.

A dense area consisting of medium-sized plants and trees frames the Fontana dei Tritoni to the southeast, while a wide green area with towering palm trees slopes gently uphill behind the fountain to the west. The palm area is completely encircled by gravel pathways.

A dense pine section lies to the north of the palm section, covering the area with large tree canopies. Within the shade, a water creek runs down a mild slope between two small lily ponds. Stepped rocks mark the path along the border of the creek. Evergreen growth characterizes the garden while continuing north in this section, and tall cypress and pine trees are planted throughout the hillside. The elevation rapidly increases as one travels the pathways leading to the northwest corner of the Orto Botanico. The northern boundary adjoining the Convento del Sacro Cuore is identified by a hedge line and dense evergreen growth.