Molecular methods offer improvement in the detection of causative pneumonia pathogens, but there are concerns of false positive results. Here we validate quantitative real-time PCR (qPCR) assays for the detection of Streptococcus pneumoniae and Haemophilus influenzae in: (a) spiked serum samples and (b) in matched serum and nasopharyngeal swabs from a population of Indigenous Australian children without pneumonia, but with a high nasopharyngeal carriage prevalence of S. pneumoniae and H. influenzae. Matched sera and nasopharyngeal swabs were selected from Indigenous children less than 5 years of age without a diagnosis of pneumonia. Specimens were assayed by qPCR targeting the lytA and glpQ genes from S. pneumoniae and H. influenzae, respectively. Using qPCR, neither S. pneumoniae nor H. influenzae DNA was detected in serum samples, even after concentration of serum DNA. In matched nasopharyngeal swabs, bacterial load was high with up to 106 cells/ml detected by qPCR. In this cohort of children with a high nasopharyngeal carriage, prevalence and bacterial load of pneumonia pathogens, qPCR on sera would not have produced a false pneumonia diagnosis. Thus, qPCR analysis of sera appears to be an appropriate method to aid aetiological diagnosis of pneumonia in this population.