Compound 29, with a rigid aromatic linker, had no antiparasitic activity at the tested concentrations, and an analogous outcome was observed for 31 presenting a short aliphatic connector. Related to 31, increased flexibility of the linker in 28 did not impact neither the effect on HFF nor the antiparasitic efficacy at 1 µM. Compounds 30 and 31 containing an ester and, respectively, an amide bond in the linker, did not impair host cells at both tested concentrations, and had no antiparasitic activity at 0.1 µM. However, ester 30 presented a stronger antiparasitic effect compared to amide 31 when applied at 1 µM, underlining the importance of the nature of the connector for the biological activity. Uracil triazole conjugates 28-31 exerted lower toxicity to the host cells compared to respective alkyne intermediates 6, 7 and 8 at 1 µM. Compared to uracil derivative 31, adenine functionalized compound 33 presented a slightly increased activity against the parasite but also higher cytotoxicity to host cells at both tested concentrations. Among the uracil conjugates 28-31, only ester analogue 30 exhibited anti-toxoplasma activity in vitro which confirms the key role of the linker upon the measured biological activity.