BACKGROUND Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples. METHODS A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively. RESULTS Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites. CONCLUSION The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.