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<i>Acanthamoeba</i>, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. <i>Acanthamoeba</i> feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that <i>Acanthamoeba</i> acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within <i>Acanthamoeba</i> following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as <i>E. coli</i>, <i>Mycobacterium</i> spp. and <i>P. aeruginosa,</i> were observed in clinical isolates of <i>Acanthamoeba</i>, whereas <i>Legionella</i>, adenovirus, mimivirus, and unidentified bacteria (<i>Candidatus</i>) were often identified in environmental <i>Acanthamoeba</i>. Increasing resistance of <i>Acanthamoeba</i> associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in <i>Acanthamoeba</i>, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of <i>Acanthamoeba</i> infections.