Yellow Canopy Syndrome (YCS) is a largely undiagnosed condition impacting sugarcane crops across Queensland, Australia, causing yield losses over 30% in severe cases. Since the first observation of YCS near Cairns in 2012, the condition has spread to all major cane growing areas and is a critical issue for the industry. Key YCS symptoms differ from leaf yellowing due to drought stress, phytotoxicity, insect attack, known diseases, nutrient deficiency or natural maturing. Despite elimination of some potential causes, the causal agent of YCS remains unknown. We aimed to determine the involvement of biotic interactions and soil nutritional health in YCS development. A microbiome-based approach using Illumina MiSeq and HiSeq was applied on 598 samples to reveal the complexity of organisms (bacteria, fungi, other eukaryotes, viruses) present in YCS-affected sugarcane and rhizosphere soil. Samples were collected from 31 cane fields growing 4 sugarcane varieties along a 600 km coastline comprising 3 climatic regions. Soil nutrient status and microbial enzyme activity were determined and did not significantly differ between affected and healthy fields. Bacterial (>25000 OTUs) and fungal (>11000 OTUs) assemblages of leaves, stalks, roots and rhizosphere soils differed between asymptomatic vs. symptomatic plants, as well as particular rhizosphere microbial functions. Community shifts with YCS appeared region- and variety specific, however, and to date few consistent bacterial and fungal signals and no consistent viral signal associated with YCS have emerged. We envision that results of this ongoing study may reveal potential causal agent(s) of YCS and/or lead to identification of specific management practices.