The timing and cause of megafaunal extinctions are an enduring focus of research interest and debate. Despite the developments in the analysis of coprophilous fungal spores (CFS), the proxy for reconstructing past megaherbivore changes, the environmental consequences of this fauna loss remain understudied. This is partly due to the general obscurity of such a signal in pollen records, as well as limitations in disentangling human and extinction ecological impact, and the lack of spatial information of megafauna changes in site-level sedimentary records. In Australia, the debate centres on the possibility that habitat loss through climate change, vegetation-fire change, human intervention, or a combination of these factors led to the extinction of some large animals during the Late Pleistocene. Pollen and plant isotope studies have also demonstrated that vegetation-fire responses following the Late Pleistocene megafaunal extinctions were characterized by increased vegetation density and fire activity due to reduced grazing/browsing pressure. Here, we use a well-dated marine sedimentary core record from the Murray Darling Basin in southern Australia and apply palynological and functional palaeoecological approaches to reconstruct the Late Pleistocene megafaunal abundance changes, the timing and potential cause of extinction across the basin and investigate if extinction was associated with any signal of trait-based vegetation changes. We infer megafaunal abundance changes from the abundance of CFS and compare this with climatic proxies from the same core. We then link modern observations of fruit, seed and fire response traits of plant genera within the basin to the fossil pollen record to reconstruct palaeo vegetation community traits and determine if extinction was associated with any changes in plant community trait composition. Closely-spaced 14C dates obtained from planktonic foraminifera and δ18O tie points place a major decline in CFS, and thus the timing of extinction, within the basin at ∼43.3 ka. While climate-driven environmental changes largely controlled megafaunal presence, human arrival and frequent landscape burning are considered the most likely primary cause of extinction or, at the very least, megafauna decline in the Murray Darling Basin. We also found that the proposed period of megafaunal decline was also accompanied and followed by a decline in the prevalence of plants with larger seeds and fruits that were likely to have been once dispersed by megaherbivores. Our study supports the idea of a human-driven megafaunal extinction in mainland Australia and that the extinction caused changes in vegetation due to reduced plant dispersal and herbivory. However, high fire activity primarily linked to these vegetation changes was not observed, as humans were already practicing landscape burning before the period of megafaunal extinction and likely continued to do so afterward.