Objective: To investigate microbial contamination in Ortho-K wearer’s lens cases caused by Staphylococcus and Pseudomonas and to solicit its risk factors. Methods: Lens cases used for at least 1 month were collected from Ortho-K wearers coming back for after-care in West China Hospital and an interview using preset questionnaire about their demographic information and lens wearing was performed. Lens cases were sampled in clean bench and the samples were then transferred onto Staphylococcus selective agar plate and Pseudomonas selective agar plate, which were incubated at 37 ℃ for 48 h before observed. Results: A total of 52 subjects were recruited, including 15 male and 37 female, with an average age of (11.8±2.5) years. Contamination rate of Staphylococcus was 42% (n=22), in which 21% (n=11) were detected with Staphylococcus Aureus (SA). With no Pseudomonas Aeruginosa (PA) was detected, the general rate of microbial contamination was 44% (n=23). Contamination rate of cases stored in living room was 25% (5/20), significantly lower than cases stored in bedroom (58%, 18/31). And cases that daily cleaning operated by parents (31%) (10/32) were less contaminated than that operated by children themselves (65%) (13/20). The increase of detection rate of microorganism in lens cases didn’t reach a significant statistical difference with longer use. Conclusion: The contamination rate of Staphylococcus, in which a considerable part was contributed by SA, is high in Ortho-K lens cases. Personnel of daily cleaning and location of case storage are the risk factors of lens case contamination. Length of case use could be a potential risk of microbial contamination but remains to be proved by further research.