Several clinical practice guidelines exist for the management of respiratory syncytial virus (RSV) infection, but the use and overuse of medications and medical tests with insufficient evidence of effectiveness remains substantial. This study aimed to evaluate the medical costs associated with bronchiolitis hospitalizations caused by RSV infection among infants aged < 2 years in Colombia. This was a prevalence-based cost-of-illness multicentric study performed from the societal perspective during 2016–2017. A case was defined as a laboratory-confirmed RSV infection with hospitalization. All costs and use of resources were collected directly from medical invoices and health records. This study included 193 patients with a diagnosis of RSV. The average hospital stay duration was 5.55 days. The major contributors to hospitalization costs were room costs (31.5%), drugs (21.8%), and indirect costs (14.9%). Medications with the highest costs were nebulization with a hypertonic solution and systemic antibiotics. In total, 96% of β-lactam antibiotics, 90% of bronchodilators, and 86% of corticosteroids and epinephrine were classified as inappropriate. RSV infection in Colombia places a high economic burden on the health system. Generating comprehensive data on healthcare resource use and costs associated with RSV will help to provide valuable information for the development of cost-effectiveness models and to guide RSV-prevention strategies.