Background Although there are several studies about the alteration in skin flora, limited number of reports about changes in the microbial contents and their resistance profile of other body sites in patients treated with isotretinoin for acne vulgaris. Objectives The aim of this study was to investigate the effects of systemic isotretinoin and antibiotic therapy on the microbial floras of oropharynx, nose and feces in acne patients. Methods Treatment groups of isotretinoin and antibiotics consisting of 20 and 15 patients, respectively were included. Microbiological culture samples were taken at baseline and once a month during 46 months of treatment period. Results Difference in microbial flora throughout the treatment period was detected at least among one of all culture samples of 15 (75%) and 5 (33%) patients in isotretinoin and antibiotic groups. There was statistically significant difference between two groups in means of alteration of the microbial flora (P=0.013). The difference was definitely observed among nasal cultures (65%) in isotretinoin group and fecal cultures (20%) in the other. Staphylococcus aureus colonization was prominent in the microbial floras of nose and oropharynx and 2 of 14 nasal isolates were detected to be methicilline resistant while Escherichia coli with extended spectrum beta lactamase activity was detected in fecal floras of patients in isotretinoin group. Conclusions Systemic isotretinoin and antibiotic treatments in acne patients precisely caused variations in the microbial floras of several sites of the body, while isotretinoin was commonly more responsible than antibiotics. Knowing that alterations in the microbial colonization of the flora regions may preceede infectious disease and bacterial resistance, treatment options and follow-up procedures in acne vulgaris should be carefully determined to reduce the risk of destruction of the microbial flora.