Aim To investigate the pathogens responsible for mastoiditis in patients, and review their antibiotic resistance at Waikato Hospital, Hamilton, New Zealand. Methodology A retrospective review to include patients who presented to Waikato Hospital with acute mastoiditis between 1/1/2018 and 31/12/2023. Patients who did not have any microbiology sample available were excluded from the study. Patient age, gender, ethnicity, co-morbidities, adverse drug reactions and allergies were recorded. Pathogens identified on microscopy, culture and sensitivity (MCS) tests of specimens including swabs, aspirates and tissue samples were recorded. Results 42 patients were included in the study, with a slight male preponderance at 52.4%. The median age was 6.5 years. 29 patients (69.0%) were aged 18 and under. Maori comprised the biggest patient group at 57.1%, followed by European at 28.6%. Streptococcus pneumoniae was the most common pathogen, identified in 8 patients (19.0%) with mastoiditis. 2 out of 8 (25%) of these were of a resistant strain. Staphylococcus aureus was the next most common in 16.7% of patients, followed by Group A streptococcus in 14.3% of patients. There was 1 (2.4%) case of MRSA. In 8 patients (19.0%), the causative pathogen was not identified. 10 patients (23.8%) had polymicrobial infections. 7 patients (16.7%) had specimen obtained for MCS prior to the administration of antibiotics. All patients were commenced on empirical antimicrobial treatment prior to the MCS results, most commonly with systemic amoxicillin-clavulanate and topical ciprofloxacin and hydrocortisone. Patients are likely to be given ceftriaxone in the context of clinical concern for intracranial complications. Conclusion Overall, the most likely causative pathogens in patients with mastoiditis are gram positive cocci. The rate of antimicrobial resistance is low, therefore amoxicillin-clavulanate remains an appropriate first-line empirical agent in uncomplicated acute mastoiditis.