Chiroptical spectroscopies provide structural analyses of molecules and nanoparticles but they require sample volumes that are incompatible with generating large chemical libraries. New optical tools are needed to characterize chirality for the ultrasmall (<1 µl) volumes required in the high-throughput synthetic and analytical stations for chiral compounds. Here we show experimentally a novel photonic effect that enables such capabilities—third-harmonic Mie scattering optical activity—observed from suspensions of CdTe nanostructured helices in volumes <<1 µl. Third-harmonic Mie scattering was recorded on illuminating CdTe helices with 1,065, 1,095 and 1,125 nm laser beams and the intensity was around ten-times higher in the forward direction than sideways. The third-harmonic ellipticity was as high as 3° and we attribute this effect to the interference of chiral and achiral effective nonlinear susceptibility tensor components. Third-harmonic Mie scattering on semiconductor helices opens a path for rapid high-throughput chiroptical characterization of sample volumes as small as 10 −5 µl.