Introduction: Duboisia leichhardtii F.Muell. is a medium to large tree which is native to subtropical regions of eastern Australia. Duboisia spp. contain a number of psychoactive tropane and pyrrolidine alkaloids with reported antibacterial activity. Despite this, D. leichhardtii leaf extracts have not been rigorously examined for growth inhibitory properties against many bacteria, including the bacterial triggers of autoimmune inflammatory diseases. Methods: The antimicrobial activity of D. leichhardtii leaf solvent extracts was investigated by disc diffusion and growth time course assays against a panel of bacterial triggers of autoimmune diseases. The growth inhibitory activity was further quantified by MIC determination and growth time course assays. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: Methanolic and aqueous D. leichhardtii leaf solvent extracts were potent inhibitors of the bacterial triggers of rheumatoid arthritis and ankylosing spondylitis. The methanolic extract displayed the most potent bacterial growth inhibitory activity. It was particularly potent against P. mirabilis (MICs of 85 and 116 µg/mL against reference and clinical strains respectively) and P. vulgaris (MIC of 187 µg/mL). The methanolic extract was also a good inhibitor of K. pneumoniae growth (MICs of 143 and 118 µg/mL against reference and clinical strains respectively). The aqueous and ethyl acetate extracts were also potent bacterial growth inhibitors, albeit with higher MIC values. The antibacterial activity of the methanolic and aqueous D. leichhardtii leaf extracts were further investigated by growth time course assays which showed significant growth inhibition in cultures of P. mirabilis and K. pneumoniae within 1 h of exposure. All extracts were determined to be nontoxic in the Artemia franciscana nauplii bioassay, indicating their safety for use in preventing these autoimmune inflammatory diseases. Conclusions: The lack of toxicity of the D. leichhardtii leaf extracts and their growth inhibitory bioactivity against the bacterial triggers of rheumatoid arthritis and ankylosing spondylitis indicate their potential in the development of new therapies targeting the onset of these diseases.