Climate change is prompting a shift of tropical pests to locations with a previously lower probability of invasion. This is the case for root-knot nematodes, Meloidogyne sp., particularly of the tropical group. Among them, M. ethiopica is now considered a threat to European food security. The development of novel sustainable nematicides can be based on in vitro bioassays of highly active phytochemicals, e.g., volatiles from essential oils. However, a steady supply of nematodes is often very difficult and dependent on environmental conditions. In the present study, an in vitro co-culture system of M. ethiopica parasitizing hairy roots of Solanum lycopersicum was established, for the first time, to easily obtain populations of second-stage juveniles (J2). These were then used to screen the nematicidal activity of 10 volatile compounds characteristic of essential oils. Finally, information on the most successful compounds was reviewed to predict their environmental dispersion and ecotoxicological hazards. The M. ethiopica population obtained from the co-culture was morphologically similar to reported populations in natural conditions and could be accurately used in direct-contact bioassays. The aldehydes citral and citronellal induced complete mortality of the tested J2, at 1 mg/mL, while compounds from other chemical groups were not as successful. In comparison to commonly used commercial nematicides, citral and citronellal were less likely to accumulate in the water environmental compartment and have lower reported toxicities compared to aquatic organisms and to mammals. Overall, in vitro co-cultures showed the potential to expedite the screening and discovery of bioactive compounds as a contribution to the development of sustainable biopesticides, as well as to lower the impacts of modern farming on agroecosystems.