Acanthamoeba spp. have always caused disease in immunosuppressed patients, but since 1986, they have become a worldwide public health issue by causing infection in healthy contact lens wearers. Amoebae of the Acanthamoeba genus are broadly distributed in nature, living either freely or as parasites, and are frequently associated with biofilms throughout the environment. These biofilms provide the parasite with protection against external aggression, thus favoring its increased pathogeny. This review aims to assess observational studies on the association between Acanthamoeba spp. and biofilms, opening potential lines of research on this severe ocular infection. A systematic literature search was conducted in May 2020 in the following databases: PubMed Central ® /Medline, LILACS, The Cochrane Library, and EMBASE ® . The studies were selected following the inclusion and exclusion criteria specifically defined for this review. Electronic research recovered 353 publications in the literature. However, none of the studies met the inclusion criterion of biofilm-producing Acanthamoeba spp., inferring that the parasite does not produce biofilms. Nonetheless, 78 studies were classified as potentially included regarding any association of Acanthamoeba spp. and biofilms. These studies were allocated across six different locations (hospital, aquatic, ophthalmic and dental environments, biofilms produced by bacteria, and other places). Acanthamoeba species use biofilms produced by other microorganisms for their benefit, in addition to them providing protection to and facilitating the dissemination of pathogens residing in them.