Background: Streptococcus pneumoniae (S. pneumoniae) is one of the most common causes of human diseases in young children. Macrolides are commonly antibiotics used for empirical treatment of community-acquired respiratory infections. The purpose of this study was to determine antibiotic resistance pattern as well as the relationship between macrolide resistance and the major mechanisms of resistance in pneumococci isolated from healthy children. Materials and Methods: In this cross-sectional study, 43 isolates of S. pneumoniae were collected from healthy children in Ardabil. Resistance pattern against tested antibiotics was determined using the disk diffusion method. The Minimum Inhibitory Concentration (MIC) of erythromycin was determined using the E-test strips. The mefA/E and ermB gene were detected in erythromycin-resistant isolates using the specific primers and Polymerase Chain Reaction (PCR) technique. Results: According to antimicrobial susceptibility testing, 74.4 % of the isolates were resistant to erythromycin, 95.3 % to penicillin, 81.3 % to co-trimoxazole, 72 % to azithromycin, 41.8 % to tetracycline, 27.9 % to clindamycin, and 16.2 % to chloramphenicol. All isolates were susceptible to levofloxacin and vancomycin. In the case of rifampin, 95.3% of the isolates were sensitive and 4.6% semisensitive. The MIC of erythromycin for resistant isolates was between 1.5 and ≥ 256 μg/ml. PCR results revealed that 100% of erythromycin-resistant isolates contained mefA/E gene and 81.25 % contained both the ermB and mefA/E genes. Conclusion: The prevalence of antibiotic-resistant strains of S. pneumoniae, especially resistance to macrolides, was high among healthy children in Ardabil. According to the results of this study, we suggest using levofloxacin, rifampin and vancomycin antibiotics as an appropriate prophylactic regimen in pneumococcal infections.