In the present study, the chemical profile and bioactive properties of the roots of turnip-rooted parsley (Petroselinum crispum spp. tuberosum) germplasm were evaluated. For this purpose, plants from seventeen parsley cultivars were grown in 6 L pots, and the obtained roots were analyzed in terms of nutritional value, chemical composition (tocopherols, sugars and organic and fatty acids) and bioactive content (antioxidant activity, phenolic compound composition and antimicrobial properties). Our results showed great variability in terms of the chemical composition and bioactive properties of root parsley germplasm. A higher fresh root yield was recorded for the common “Root parsley” common variety (164 g/pot), followed by the varieties “Osborne” (109 g/pot), “Sonata” (104 g/pot), “Kaśka” (104 g/pot) and “Halblange Berlinska” (103 g/pot), whereas the lowest yield was recorded for the “Hanacka” variety (69 g/pot). A significant variation was also observed in the nutritional value parameters: the roots of the “Sonata” genotype showed the highest fat content; “Arat”, “Osborne” and “Olomuńcka” had the highest ash content; the “Alba” cultivar contained significantly higher amounts of carbohydrates; and the “Vistula” cultivar showed the highest energetic value. The only detected isoforms of vitamin E were α- and δ-tocopherols; content varied depending on the cultivar, although α-tocopherol was the most abundant compound in most cultivars, especially in the “Arat” cultivar. Sucrose was the most abundant free sugar detected, especially in the “Sonata” cultivar (16.96 g/100 g dw), followed by apiose (2.93–5.55 g/100 g dw), glucose (1.3–3.47 g/100 g dw) and fructose (1.37–3.03 g/100 g dw). Moreover, malic acid was the most abundant organic acid in most of the tested cultivars. Twenty-one individual fatty acids were identified in all the studied cultivars, with linoleic (47.9–57.1%) and palmitic acid (20.66–20.5%) being the most abundant. Nineteen individual phenolic compounds were tentatively identified, including three phenolic acids, fourteen flavonoids and two hydrolyzable tannins, while apigenin-O-pentoside-O-hexoside was the most abundant. The antioxidant activity differed between the tested assays (TBARS and OxHLIA), and the most effective cultivars for the TBARS assay (“Root parsley (common variety)” and “Berlinski Halblange Springer”) were those with the lowest antioxidant activity for the OxHLIA assay after 120 min. Finally, in most cases, the root extracts were more efficient or similarly effective compared to the positive controls against the tested bacteria and fungi. In conclusion, our results provide information regarding the chemical characterization and the bioactivities of the roots of turnip-rooted parsley germplasm that could be further exploited in sustainable and diversified agro-ecosystems through the introduction of this species as a novel/complementary crop in the traditional farming systems of the Mediterranean basin.