Radopholus similis (Cobb 1893) (Rhabditida, Pratylenchidae) is a common and destructive pest that causes significant yield losses in Cavendish bananas. The repeated use of synthetic pesticides to control this nematode has raised concerns about toxicity to users and the environment. An alternative approach to managing this harmful species involves the use of nontoxic plant-derived extracts that enhance protection against various biological constraints. In this study, aqueous extract from the leaves and stems of Eupatorium odoratum Linn. (EAEO) was used to control R. similis in banana roots under in vitro and on-plant conditions. Through direct contact and penetration experiments on roots, as well as on-plant trials, concentrations of 12500, 6250, 3120, 1560, and 780 mg DW·L−1 EAEO were used to evaluate nematode mortality rates, infection levels in root segments (in vitro experiment), root infection, root necrosis index, shoot and root fresh weight, and growth parameters such as stem length, diameter, leaf number, and leaf surface area (on-plant experiment). The results showed that the nematode mortality rate due to EAEO ranged from 35.3% to 90.1%, which is statistically significant compared to the mortality rate in the control treatment (16%). Significant nematode inhibition was observed in banana roots infected with R. similis and exposed to EAEO for 48 h. There were no statistically significant differences in root infection, root necrosis, and plant fresh weight between plants treated with fenamiphos and EAEO. Maximum effectiveness was observed at the 12500 mg DW·L−1 EAEO concentration. These findings provide valuable insights into the nematicidal metabolites of EAEO against R. similis. Thus, the potential of EAEO as a biocontrol agent against R. similis has been demonstrated. For future research, investigations will be extended to elucidate the mechanisms of action of the essential compounds in EAEO against nematodes, determine the optimal EAEO dosage for nematode control without harm to banana plants and the environment, assess the long-term effects of EAEO on banana plants and the environment, explore the ecological impact of EAEO use in agricultural ecosystems, study the effectiveness of EAEO on various plant species, and consider the development of commercial products or formulations based on EAEO.