Background: To investigate bacterial flora and antibiotics susceptibility of Streptococcus pneumoniae isolated from the conjunctival sac of heathy children. Methods: Bacteria were isolated from the secretions of conjunctival sac of healthy children between 2015 and 2018. Antimicrobial susceptibility of isolated S. pneumoniae strains were determined using microbroth dilution method. Results: The sac secretions were collected from a total of 6440 children. 1409 samples presented bacterial growth, accounting for 21.8% of the samples. Among the 22 bacterial species isolated, 528 samples presented Grampositive Staphylococcus spp. growth, accounting for 37.4% of the isolates, followed by Corynebacterium spp., counting for 30% of the isolates and Streptococcus pneumoniae, counting for 21.4% of the isolates. Antibiotics susceptibility tests showed that the majority of S. pneumoniae isolates were sensitive to most antibiotics tested. However, 72.8 and 81.2% of the isolates were resistant to erythromycin and tetracycline, respectively, and over 10% of them were resistant to gentamicin, tobramycin and rifampicin. Conclusions: The bacterial flora of healthy children is mainly consisted of Gram-positive bacteria belonging to Corynebacterium spp. and Streptococcus spp.; most of S. pneumoniae isolates were sensitive to antibiotics except erythromycin and tetracycline.