The third section of the Orto Botanico stretches steeply up the Janiculum Hill toward the statue of Garibaldi, and covers the western most portion of the garden. The southern segment of this area features a spectrum of organized specialty gardens. Naturalized vegetation overgrowth and heavily forested areas are characteristics of the remaining portion of the third section. The Scala d'Acqua is situated in the central portion of this section, leading to the Nicchione located outside the western boundary of the Orto Botanico.

The two paths of the third section meet at the northwest corner of the Orto Botanico and create a walkway to the Roman Woodland. The woodland contains remnants of the deciduous forest that once covered this area of Rome.

A Japanese Garden is located in the southwest corner of the Orto Botanico. This garden is composed of an organized series of minor paths, waterways, and topiary. A pagoda overlooks small ponds and provides a clear vista of Rome's skyline.

Beyond the Japanese Garden, a winding path leads up the hill to a closed entrance of the Orto, located off of Viale del Parco di Villa Corsini. Although this entrance is no longer in use, the gate house remains and an observation landing provides another view of the city. A collection of irises, comprised of potted urns planted in the steep slope, is located between the observation landing and the upper path. This area leads into the Rock Garden, which is situated below the irises on the western most path. A small stream weaves throughout the area and empties into a larger pond.

As in the northwest corner, the two paths merge at the bamboo forest that was planted in the early 1990's. Over 150 species grow successfully in the garden. However, bamboo is a very invasive species, and has overwhelmed both sides of the path, creating a tunnel effect. Continuing east, the visitor reaches the Fern Garden. An internal network of paths provides access to the Fern Garden and planted beds slope steeply on both sides. A small stream winds through the ferns.

The Rose Garden is the last thematic garden in the third section and is northwest of the Fern Garden. A rose trellis creates the western boundary of the Rose Garden. Continuing north, the remaining portion is covered by moderately dense vegetation.