Plant-parasitic nematodes cause substantial damage to agricultural crops worldwide. Long-term management of these pests requires novel strategies to reduce infection of host plants. Disruption of nematode chemotaxis to root systems has been proposed as a potential management approach, and novel assays are needed to test the chemotactic behavior of nematodes against a wide range of synthetic chemicals and root exudates. Two microfluidic chips were developed that measure the attraction or repulsion of nematodes to chemicals (“chemical chip”) and young plant roots (“root chip”). The chip designs allowed for chemical concentration gradients to be maintained up to 24 h, the nematodes to remain physically separate from the chemical reservoirs, and for images of nematode populations to be captured using either a microscope or a flatbed scanner. In the experiments using the chemical chips, seven ionic solutions were tested on second-stage juveniles (J2s) of Meloidogyne incognita and Heterodera glycines. Results were consistent with previous reports of repellency of M. incognita to a majority of the ionic solutions, including NH4NO3, KNO3, KCl, MgCl2, and CaCl2. H. glycines was found to be attracted to both NH4NO3 and KNO3, which has not been reported previously. A software program was written to aid in monitoring the location of nematodes at regular time intervals using the root chip. In experiments with the root chip, H. glycines J2s were attracted to roots of 3-day-old, susceptible (cultivar Williams 82) soybean seedlings, and attraction of H. glycinesto susceptible soybean was similar across the length of the root. Attraction to resistant (cultivar Jack) soybean seedlings relative to the water only control was inconsistent across runs, and H. glycines J2s were not preferentially attracted to the roots of resistant or susceptible cultivars when both were placed on opposite sides of the same root chip. The chips developed allow for direct tests of plant-parasitic nematode chemotaxis to chemicals and roots with minimal human intervention.