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Genetically uniform plant material, derived from <i>Lychnis flos-cuculi</i> propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic <i>Acanthamoeba castellani</i>. Additionally, a Microtox^® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC₅₀ of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC₅₀ of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25–2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of <i>L. flos-cuculi</i> in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from <i>L. flos-cuculi</i> micropropagated, ecdysteroid-rich plant material.