OBJECTIVE Intravenous therapy is a complex procedure usually requiring the preparation of the medication in the clinical area before administration to the patient. Breaches in aseptic technique may result in microbial contaminations of vials which is a potential cause of different avoidable infections. We aimed to investigate the prevalence and pattern of microbial contamination of single- and multiple-dose vials in the largest pulmonary teaching hospital in Iran. METHODS In a period of 2 months, opened single- and multiple-dose vials from different wards were sampled by a pharmacist. The name of the medication, ward, labeling of the vials, the date of opening, and storing temperature were recorded for each vial. Remained contents of each vial were cultured using appropriate bacterial and fungal growth media. RESULTS Microbial contamination was identified in 11 of 205 (5.36%) of vials. The highest contamination rate was 14.28% for vials used in interventional bronchoscopy unit. The most frequent contaminated medication was insulin. Gram-positive bacteria (81.82%) were more significantly involved than gram-negative ones (9.09%) and fungi (9.09%), with the highest frequency for Staphylococcus epidermidis. CONCLUSIONS Our data demonstrate that repeated use of vials especially if basic sterility measures are disobeyed can cause microbial contamination of administered products to the patients. Infection preventionists are responsible to train health care workers regarding aseptic techniques and apply guidelines for aseptic handling of intravenous solutions.