Streptococcus pneumoniae (pneumococcus) is recognized as one of the major cause of infections in communities and hospitals. In this study, anti-pneumococcal and anti-efflux pump activity of two medicinal plants (Thymus daenensis and Origanum vulgare) essential oils were evaluated. Checkerboard assay test was performed for investigation of the effects of selected EOs on ciprofloxacin (CIP) and ethidium bromide (EtBr) uptake in pmrA-overexpressed fluoroquinolone-resistant pneumococcus. Using quantitative real-time RT-PCR the PmrA efflux pump gene (pmrA) expression was evaluated following treatment with selected EOs. Gas chromatography-mass spectrometry analysis was performed for identifying the major components of the tested EOs. The minimum inhibitory concentration (MIC) values for pneumococcus isolates were 0·625-2·5 μl ml for T. daenensis and 1·25-5 μl ml for O. vulgare EOs. We confirmed that in all strains T. daenensis and O. vulgare have a total or partial synergistic effects with CIP and EtBr (FICI from 0·14 to 0·75). In other hand MIC/2 concentration of T. daenensis and O. vulgare EOs caused a significant downregulation of pmrA gene (P < 0·05) in seven of eight strains. This study showed that T. daenensis and O. vulgare EOs have strong antimicrobial and anti-efflux pump activity against clinical isolates of S. pneumoniae and might be useful in controlling pneumococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduced Thymus daenensis and Origanum vulgare essential oil as new antibacterial and anti-efflux pump agents against fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates. These findings indicate that combination of these two essential oils with fluoroquinolone antibiotics may provide alternative methods to overcome the fluoroquinolone-resistant S. pneumoniae.