Background: Atopic Dermatitis (AD) is a chronic relapsing, pruritic inflammation of the skin which is often colonized by Staphylococcus aureus. Antibiotic resistance of S. aureus is a constant challenge for clinicians who manages atopic dermatitis. Aim: To determine S. aureus antibiotic resistance pattern among patients with non-infected atopic dermatitis and its association with disease severity. Methods: One hundred and seventy eight participants (89 AD patients and 89 controls) were recruited from Universiti Malaya Medical Centre (UMMC). Participants were subjected to a questionnaire on demographics, personal and family medical conditions as well as antibiotic administration. AD severity were determined using Scoring Atopic Dermatitis (SCORAD). Skin swab was taken from eczematous lesion in patients and from left forearm in controls. Antibiotic susceptibility towards methicillin, vancomycin, rifampicin, fusidic acid, erythromycin, gentamicin, clindamycin, sulphamethoxazole, cefuroxime and penicillin were determined using disk diffusion method. Results for antibiotic resistance were categorized as none, sensitive and resistant. Results: Colonization of S. aureus in AD were significantly higher than control (p<0.001). Highest antibiotic resistance was reported for Penicillin (32/39, 82.1%), followed by Fusidic Acid (7/39, 17.9%) as well as Clindamycin and Erythromycin (3/39, 7.7% respectively). Two AD patient (5.1%) were resistant to Gentamicin. In addition, 1 AD patient (2.6%) was resistant towards Methicillin, Sulfamethoxazole and Cefuroxime respectively. No antibiotic resistance was reported for Vancomycin and Rifampicin among the AD patients. Conclusion: High resistance were found for Penicillin and Fusidic acid. Their usage and prescription should be reduced to preserve its sensitivity.