The role of biotic agents in the dieback syndrome Mundulla Yellows (MY) was investigated by analysis of 40 Eucalyptus camaldulensis, E. leucoxylon, or E. cladocalyx trees and soil samples from South Australia and Victoria, Australia. No pathogenic fungi, bacteria, phytoplasmas, or insect pests or vectors were found to be associated with MY. However, nematode analysis identified Merlinius spp. to be associated with soil, but not roots, from symptomatic trees. Interveinal chlorosis symptoms were not transmissible by seed, mechanical inoculation, or grafting using plant material derived from symptomatic trees. Virus-like particles were detected at a single symptomatic study site using transmission electron microscopy. MY symptoms were induced in E. camaldulensis seedlings by sowing seed from asymptomatic trees into sterilized and unsterilized soil collected from underneath symptomatic trees. Significantly, sterilized soil induced more severe symptoms in seedlings than unsterilized soil. Soil collected from under asymptomatic trees did not induce MY symptoms. This preliminary investigation indicates that, with the exception of Merlinius spp., pathogenic organisms and pests were not consistently associated with MY symptoms.