Introduction Mastoiditis resulting from acute otitis media is a serious complication mainly affecting children. Intracranial abscess and sigmoid sinus thrombosis may arise, resulting in significant morbidity. Successful treatment requires hospitalisation, surgical intervention, and targeted antibiotic use. Understanding local microbiological trends is critical to guide suitable antibiotic therapy. Aims This study analysed the microbiology of paediatric mastoiditis in Queensland and its correlation with complications. Susceptibility to current recommended empirical therapy was assessed. Methods A retrospective case series reviewed all paediatric patients presenting with acute mastoiditis who underwent operative management between 2015 and 2024 at the two tertiary paediatric centres in Queensland – Queensland Children’s Hospital and Townsville University Hospital. Demographics, clinical data, microbiology, antibiotic sensitivities, and complications were evaluated. Results Over a ten-year period, 263 patients showed a statistically significant increasing incidence of surgically managed acute mastoiditis. The most frequently isolated organisms were Streptococcus pneumoniae (24%, n=62) and Streptococcus pyogenes (14%, n=37). Cultures from 23% (n=61) demonstrated no bacterial growth, likely reflecting prior antibiotic use or difficulty in culturing bacteria. Of 138 positive cultures, only 4% (n=5) had proven resistance to cefotaxime. Complicated mastoiditis was seen in 18% (n=48) of patients. Fusobacterium species were strongly associated with complications (75%, p<0.01) and predominantly required 16sRNA for identification (n=5, 63%), after being culture negative initially. Conclusions The microbiology of acute paediatric mastoiditis in Queensland is broadly consistent with international trends. Fusobacterium species are a fastidious organism most strongly associated with complications. The local empirical antibiotic recommendation of cefotaxime remains appropriate.