Festulolium hybrids are cool-season forage grasses that form symbiotic relationships with the fungus Epichloë uncinata, which produces loline alkaloids that protect the host from herbivores. This study evaluated the nematotoxicity of shoot and root extracts of Festulolium loliaceum against the stubby root nematode Trichodorus primitivus. Methanolic root and shoot extracts from plants aged 8, 12, 16, and 20 weeks were tested in vitro at five concentrations (312.5–5000 µg mL⁻¹) over 24, 48, and 72 h. Nematode immobility, mortality, and phytochemical profiles, including flavonoids, loline alkaloids, and phenols, were quantified. Extracts from shoots caused significant concentration and time-dependent immobility of T. primitivus (p = 0.001), reaching ≥90% at 5000 µg mL⁻¹ after 72 h in 8–12-week-old plants. Endophyte presence enhanced nematotoxicity, where LD₅₀ values for E+ roots were two-fold lower at 12 weeks and fifty-fold lower at 20 weeks compared with E− root extracts. Shoot extracts of E+ grass had the highest nematicidal activity at 8 weeks, with a significantly lower LD₅₀ value than E− (p < 0.05). Loline alkaloid concentrations increased with plant age, while flavonoids and phenols declined. Nematotoxicity of F. loliaceum extracts was strongly influenced by plant age and endophyte presence. Younger E+ shoots produced the most potent shoot extracts, whereas older plants produced the most potent root extracts. Flavonoid content was negatively correlated with shoot biomass (R = −0.94, p < 0.001). Similarly, phenol content was negatively correlated to both root biomass (R = −0.79, p < 0.001) and shoot biomass (R = −0.67, p < 0.005).