Background: Amoebae of the genusAcanthamoebacause a sight-threatening infection calledAcanthamoebakeratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains. Methods: Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored. Results: In a phase-contrast microscope,Acanthamoebasp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond toA. mauritanensis,A. culbertsoni,A. castellanii, genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites’ long duration ability to intense multiply. Conclusions: Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successiveAcanthamoebastrains with a long duration of high dynamics.