The present study describes two cases of Acanthamoeba infections (keratitis and ascites/peritonitis) in small breed domestic dogs in Tenerife, Canary Islands, Spain. In both cases, amoebic trophozoites were observed under the inverted microscope and isolated from the infected tissues and/or fluids, without detecting the presence of other viral, fungal or bacterial pathogens. Amoebae were isolated using 2 % non-nutrient agar plates and axenified for further biochemical and molecular analyses. Osmotolerance and thermotolerance assays revealed that both isolates were able to grow up to 37 °C and 1 M of mannitol and were thus considered as potentially pathogenic. Moreover, the strains were classified as highly cytotoxic as they cause more than 75 % of toxicity when incubated with two eukaryotic cell lines. In order to classify the strains at the molecular level, the diagnostic fragment 3 (DF3) region of the 18S rDNA of Acanthamoeba was amplified and sequenced, revealing that both isolates belonged to genotype T4. In both cases, owners of the animals did not allow any further studies or follow-up and therefore the current status of these animals is unknown. Furthermore, the isolation of these pathogenic amoebae should raise awareness with the veterinary community locally and worldwide.