AbstractContamination of nano‐biothreats, such as viruses, mycoplasmas, and pathogenic bacteria, is widespread in cell cultures and greatly threatens many cell‐based bio‐analysis and biomanufacturing. However, non‐invasive trapping and removal of such biothreats during cell culturing, particularly many precious cells, is of great challenge. Here, inspired by the wake‐riding effect, a biocompatible opto‐hydrodynamic diatombot (OHD) based on optical trapping navigated rotational diatom (Phaeodactylum tricornutumBohlin) for non‐invasive trapping and removal of nano‐biothreats is reported. Combining the opto‐hydrodynamic effect and optical trapping, this rotational OHD enables the trapping of bio‐targets down to sub‐100 nm. Different nano‐biothreats, such as adenoviruses, pathogenic bacteria, and mycoplasmas, are first demonstrated to be effectively trapped and removed by the OHD, without affecting culturing cells including precious cells such as hippocampal neurons. The removal efficiency is greatly enhanced via reconfigurable OHD array construction. Importantly, these OHDs show remarkable antibacterial capability, and further facilitate targeted gene delivery. This OHD serves as a smart micro‐robotic platform for effective trapping and active removal of nano‐biothreats in bio‐microenvironments, and especially for cell culturing of many precious cells, with great promises for benefiting cell‐based bio‐analysis and biomanufacturing.