The accurate diagnosis of lower respiratory tract infection remains a challenge, with conventional diagnostic methods often failing to identify a causative agent. Here we describe a multiplex real-time PCR assay that has been validated for the detection of the rarely identified atypical bacterial pathogens: Legionella pneumophila, Mycoplasma pneumoniae, and Chlamydophila pneumoniae. Due to the complexity and poor performance of the existing cultural and serological methods for the detection of these pathogens, investigation for them is rarely initiated. This is likely to result in underdetection and empirical treatment regardless of a microbiological diagnosis. The assay described here is designed to address this need.