Respiratory viral and atypical bacterial agents lead to infections in a large spectrum, from mild symptoms to respiratory failure. In the present study, we aimed to detect multiple viral and bacterial agents in the respiratory samples of inpatients by real-time polymerase chain reaction (RT-PCR). Nasopharyngeal swabs and broncho-alveolar lavage samples from inpatients with respiratory infection symptoms at the Uludag University Hospital between December 1, 2015 and March 31,2018 were investigated. DNA/RNA was extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Belgium) with the EZ1 extraction device (Qiagen, Belgium). The R-GENE (R) RT-PCR (Biomerioux, France) kit was used to detect influenza A, influenza B, respiratory syncytial virus (RSV), human metapneumovirus, rhinovirus/enterovirus (RV/EV), adenovirus, human bocavirus (hBoV), corona virus, parainfluenza virus, Chlamydia pneumoniae/Mycoplasma pneumoniae, and Legionella pneumophila in Rotor-Gene Q (Qiagen, Belgium). Patients were aged between 0 and 90 years. Overall, 177 (56.9%) patients were men and 134 (43.1%) were women. A total of 311 samples were analyzed, of which 214 (68.8%) were positive. In total, 360 agents, including 338 viruses and 22 bacteria, were detected. The commonest agents were influenza A+B (n = 65, 18,1%), hBoV (n = 64, 17.8%), RV/EV (n = 56, 15.6%), and RSV (n = 47, 13.1%). Rapid diagnosis of viral infections by RT-PCR is important for the specific treatment of patients.