IntroductionEmpyema is one of the important causes of pediatric hospital admissions.AimIn this study, we had investigated the frequency rates of S aureus, S pneumoniae, H influenzae, and P aeruginosa using PCR and bacterial culture among children suffering from empyema in Ahvaz city, Iran.MethodsThis was a descriptive study conducted on the patients hospitalized in ICUs of two teaching Hospitals of Ahvaz, Iran, between March and September 2018 on 105 pleural fluid (PF) samples of the children less than 16 years of age with the diagnosis of empyema thoracis. These specimens were inoculated on the bacterial culture media and identified using biochemical characteristics. Then, the existence of the four pathogens mentioned above was evaluated using PCR method.ResultIn this study, these bacteria agents were identified in 81 (77.14%) and 30 (28.57%) cases using the PCR assay and bacterial culture, respectively. Moreover, the PCR assay identified the infectious agents in 51 (68%) of PFs where the culture method failed. S pneumoniae (63 cases) was recognized as the most common pathogen, followed by P aeruginosa(19 cases), S aureus(15 cases), and H influenzae (9 cases) using the bacterial culture and PCR. Co‐infections were detected in 21 samples (20%) using PCR and one sample using the bacterial culture (P aeruginosa and S pneumoniae).ConclusionIn this study, we found the higher frequencies of these microorganisms using PCR than culture. In addition, we showed that PCR was a sensitive and accurate method that unaffected by antibiotic therapy and could detect well co‐infections.