The increasing use of nanomaterials in almost all sectors of society (e.g., health or energy to agriculture and transport) has generated a need for innovative detection methods for nanomaterials, to enable their continued development, environmental and toxicological monitoring, and risk assessment. In vivo nanoparticle visualization is needed to support applications in drug delivery to plant biology where real-time monitoring is essential. Techniques are sought that do not require the addition of molecular tags or nanotags to enhance detection, because these may modify the surface properties or behavior of the nanomaterials. Here two-photon excitation microscopy coupled with plant, nanomaterial, or chemical autofluorescence is used to detect and visualize multiwalled carbon nanotubes (MWCNTs), titanium dioxide, and cerium dioxide in living wheat tissues. The potential of the technique to track chemical−nanomaterial interactions in living tissues is then demonstrated, using phenanthrene as a model compound. MWCNTs were observed to pierce wheat root cell walls and enhance the transport of phenanthrene into the living cells. The ability of this technique to monitor real-time in vivo nanomaterial behavior and its potential applications and limitations for use in various disciplines is highlighted.