Cryptosporidium species are major pathogens severely affecting the health of neonate animals, in particular calves, but also cause life-threatening infection of immunocompromised humans. Currently, only halofuginone is approved for prophylactic and metaphylactic treatment of calves but this drug suffers from its limited safety margins. For treatment of immunodeficient human patients, only nitazoxanide and paramomycin are used but data regarding their efficacy are controversial. Aim of the present study was to test a substituted benzimidazole (BAY-AF76184) and a heterocyclic substituted 1,2,4-triazin (BAY-AB24992), both drugs with known anti-protozoal activity, for their potential ability to interfere with Cryptosporidium parvum development in vitro. Development of C. parvum in HCT-8 cells treated with these compounds was compared to negative controls and parasites treated with paromomycin or halofuginone using real-time PCR targeting the 18S rDNAs of parasites and host cells as template. Potential cytotoxic and anti-proliferative effects of drugs were analysed using a lactate dehydrogenase and a WST-1 assay, respectively. BAY-AF76184 and paromomycin dose-dependently inhibited development of C. parvum with EC50 values of 2.37 μM and 69.5 μM, respectively. Although high concentrations of halofuginone and BAY-AB24992 also significantly inhibited parasite development, effects of lower concentrations were very heterogeneous preventing calculation of EC50 values. Halofuginone and BAY-AF76184 dose-dependently interfered with host cell proliferation (EC50 values of 0.35 μM and 9.07 μM, respectively) and the latter also had direct cytotoxic effects (EC50=48.78 μM). However, drug concentrations required for cytopathic were higher than those for effects against C. parvum. Therefore, both BAY-AB24992 and BAY-AF76184 should be considered for further evaluation, e.g. using in vivo models.