Acute otitis media (AOM) is occasionally caused by Eustachian tube dysfunction and obstruction; it is second most common disease in children. The objective of this study is to determinate and isolate the causative agents of AOM among children and antimicrobial susceptibility pattern to certain antibiotics. A cross sectional hospital based study on 100 acute otitis media patients seen in Khartoum Ear Nose throat Teaching hospital. Patients' information was collected using pre-structural questionnaire. Ear swabs were collected from discharging ears and processed for bacteria culture and susceptibility to different antimicrobial agent using standard microbiological techniques. Out of 100 ear swabs collected and processed through standard microbiological techniques, 68 (total number of culture plate) were culture positive and 32 were negative for bacterial growth. The most common isolated microorganism was Staphylococcus aureus 38 (from total number of isolated organism = 78) (48.7%), followed by Pseudomonas aeruginosa 15 (19.2%), Escherichia coli 4 (5.1%), Citrobacter freundii 8 (10.3%), Morgenalla morganii 9 (11.5%) and Edwardsiella tarda 4 (5.1%). All isolates were resistant to Ampicillin (100%). Almost all isolated bacteria were sensitive to two or more antibiotics. About 6 (15.8 %) of S. aureus were resistant to Methicillin. In conclusions, S. aureus was the major isolated bacteria, about (15.8%) of S. aureus are Methicillin resistance that cause acute otitis media. Ciprofloxacin, Ceftazidime, Gentamicin and Ceftriaxone were relatively effective antibiotics. However, all isolates showed multi drug resistance to the Ampicillin.