Prickly acacia (Vachellia nilotica subsp. indica, Family: Fabaceae) is an invasive woody weed in coastal and semi-arid rangelands of Australia. A prominent dieback event was observed on this species in 2010 in north-western Queensland. A Botryosphaeriaceae fungus, Cophinforma sp., was consistently isolated from symptomatic stem tissues. In preliminary studies, Cophinforma sp. and an isolate of Lasiodiplodia pseudotheobromae sourced from dieback-affected Parkinsonia aculeata (Family: Fabaceae) were found to be pathogenic to prickly acacia seedlings and juveniles. In this study, we investigated whether typical dieback symptoms could be replicated under glasshouse conditions and in the field following stem inoculations of these two fungi (either singly or in combination). In the glasshouse trial, stem lesions and leaf mortality were observed following stem inoculation by both of the test fungi although it was greatest in the presence of L. pseudotheobromae. However, no effects on plant growth or mortality were observed. In the field trials (located in central and north-western Queensland) both test fungi caused infection, but significant lesions were only induced by L. pseudotheobromae in central Queensland, and no treatment effects on plant growth or survival were observed at either site over the next two years. Prominent decline in plant vigour was observed in north-western Queensland two years after inoculation, but was presumed to be naturally occurring as it affected controls and neighbouring untreated plants equally. The test fungi were reisolated from lesions in both field and glasshouse trials, but were never found in adjacent tissue, suggesting that infection was successfully contained by the plant’s wound response. We found no potential for the tested fungal isolates to be effective biocontrol agents, although future studies should aim to initiate systemic infections.