Background This study aimed to evaluate the occurrence of Streptococcus pneumoniae and Haemophilus influenzae in sputum of patients with community-acquired pneumonia (CAP) using culture and multiplex polymerase chain reaction (M-PCR) methods and to survey the antibiotic resistance patterns of aforesaid isolates. Result In total, 23.9 % (n = 22/92) of sputum samples showed positive results in the culture method. S. pneumoniae and H. influenzae were isolated from 15 (16.3 %) and 7 (7.6%) samples, respectively. Using M-PCR, 44 (47.8 %) samples were positive for S. pneumoniae and H. influenzae. Of these, S. pneumoniae and H. influenzae were detected in 33 (35.8%) and 11 (11.9%) of the sputum samples, respectively. The sensitivity, specificity, and accuracy rates of PCR in detection of S. pneumoniae in comparison with culture method were 100, 76.6, and 83.6%, respectively. While, the sensitivity, specificity, and accuracy rates of PCR in detection of H. influenzae in comparison with culture method were 100, 95.3, and 95.8%, respectively. Out of 11 isolates of H. influenzae, two strains confirmed as H. influenzae type b (Hib) and 3 isolates were type f. However, 6 isolates were non-typable. The co-trimoxazole and amoxicillin/clavulanate were the less effective antibiotics against S. pneumonia and H. influenzae, respectively. Ceftriaxone with 13.3% resistance rates was the most effective antibiotic against S. pneumoniae, while, clarithromycin, ceftriaxone, and gentamicin with resistance rates of 28.6% for each one were the most effective chemicals against H. influenzae isolates. Conclusion In this study, the prevalence of S. pneumoniae was more than H. influenzae using culture and M-PCR methods. The M-PCR provided better efficiency in detecting the bacterial agents in CAP patients compared to culture method. This method can improve the early detection of pathogens contributed to CAP. The drug resistant S. pneumoniae and H. influenzae indicated the need to develop a codified monitoring program to prevent further spread of these strains.

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