Prokaryotic Argonautes (pAgos) are an emerging class of programmable nucleases that are considered to be more flexible than existing CRISPR–Cas systems, showing great potential for biotechnology. The current applications of pAgos mainly include molecular diagnostics and in vitro DNA assembly. However, early research primarily focused on thermophilic pAgos. To enable in vivo biotechnological applications such as genome editing, researchers have gradually shifted their focus to mesophilic organisms. Here, this review will discuss the known pAgos and their various applications, as well as the challenges and potential strategies for applying pAgos in prokaryotic and eukaryotic organisms.