Amoebae serve as a critical training ground for microbial pathogens, facilitating horizontal gene transfer between amoebae and their prey, intracellular microbes, endosymbionts, and giant viruses. This interaction has shaped the evolution, selection, and adaptation of microbes that evade amoeba predation and infect mammals. Many pathogens, including Legionella, Chlamydia, and Rickettsia, have evolved to exploit amoebae as environmental hosts, utilizing their intracellular environment for survival and replication. These pathogens have acquired eukaryotic-like proteins and domains, such as ubiquitin-related proteins and tandem repeats, which are absent in mammalian cells. Amoebae and mammalian cells may respond similarly to microbial factors targeting conserved eukaryotic processes, but mammalian cells may inadvertently respond to amoeba-adapted factors, leading to accidental immune responses. Amoebae provide a unique microcosm for multi-directional horizontal gene transfer, enabling the evolution of pathogens with specialized tools to infect mammals. The co-evolution of pathogens with amoebae has enhanced their ability to survive and replicate within mammalian cells, leading to life-threatening infections. The genetic diversity of amoebae and their intracellular symbionts represents a rich source of genetic material, contributing to the evolution of microbial pathogens. The study of amoebae-microbe interactions is crucial for understanding the emergence of pathogens and their adaptation to mammalian hosts. Future research should focus on genomic, biochemical, and cellular data of unicellular eukaryotes to further elucidate these interactions and their implications for pathogenesis.Amoebae serve as a critical training ground for microbial pathogens, facilitating horizontal gene transfer between amoebae and their prey, intracellular microbes, endosymbionts, and giant viruses. This interaction has shaped the evolution, selection, and adaptation of microbes that evade amoeba predation and infect mammals. Many pathogens, including Legionella, Chlamydia, and Rickettsia, have evolved to exploit amoebae as environmental hosts, utilizing their intracellular environment for survival and replication. These pathogens have acquired eukaryotic-like proteins and domains, such as ubiquitin-related proteins and tandem repeats, which are absent in mammalian cells. Amoebae and mammalian cells may respond similarly to microbial factors targeting conserved eukaryotic processes, but mammalian cells may inadvertently respond to amoeba-adapted factors, leading to accidental immune responses. Amoebae provide a unique microcosm for multi-directional horizontal gene transfer, enabling the evolution of pathogens with specialized tools to infect mammals. The co-evolution of pathogens with amoebae has enhanced their ability to survive and replicate within mammalian cells, leading to life-threatening infections. The genetic diversity of amoebae and their intracellular symbionts represents a rich source of genetic material, contributing to the evolution of microbial pathogens. The study of amoebae-microbe interactions is crucial for understanding the emergence of pathogens and their adaptation to mammalian hosts. Future research should focus on genomic, biochemical, and cellular data of unicellular eukaryotes to further elucidate these interactions and their implications for pathogenesis.