Acanthamoeba is an opportunistic protozoan pathogen which is found in diverse environment worldwide. Being ubiquitous nature of this amoeba we come across it in our daily life. Acanthamoeba species are recognized as human pathogens; that may cause blinding keratitis and rare but fatal granulomatous encephalitis involving central nervous system. To date, there is not a single report in literature demonstrating anti-Acanthamoeba antibodies among the Saudi population, and thus aim of the present study. Using ELISA, we identified the antibody level in the local population. Our results represent the secretory IgA antiAcanthamoeba in mucosal secretions from 133 individuals aged 15–60 years. The antiAcanthamoeba antibody prevalence rate was > 80%, and no considerable differences were observed between prevalence in males (80.28%) and that in females (80.64%). In addition, environmental sources (soil and water) from the environment of the participants in our study were evaluated for amoeba incidence. The amoeba was identified by morphological characteristics of cysts or trophozoites on non-nutrient agar plates grown with E. coli. Overall, 58.75% of samples from water and 32.85% of those from soil were culture positive for outgrowth of amoeba on non-nutrient agar plates. Furthermore, PCR was carried out with genus-specific primers to confirm the presence of Acanthamoeba DNA. Our results revealed that about 68% of cultures from water and 43% of those from soil were successfully amplified and proved to be amoeba DNA. Interestingly, a few samples yielded more than one product, which suggests that some other amoebic species may be present in the same sample (MAC-W1 and MADW1). To the best of our knowledge, we described for the first time the amoeba isolation from the participant’s close environment and antibodies level among Saudi population. Our future studies will be focused on additional molecular characterization of isolated amoeba and their pathogenic potential which could be a possible threat for the community.