Background <p>Antibiotic use not only can treat chlamydia infection but may also have an impact on chlamydia transmission. This study assessed whether prior antibiotic use was associated with a lower prevalence of chlamydia infection among women in a country with high antibiotic consumption.</p> Methods <p>A cross-sectional study was conducted in 2019 among sexually active women aged 18–44 years in Guangdong Province, China. Participants seeking gynecological care underwent current chlamydia and gonorrhea testing using nucleic acid amplification. Antibiotic use in the past year was self-reported. Logistic regression models assessed the association between prior antibiotic use and the prevalence of chlamydia infection.</p> Results <p>This study surveyed 391 participants (average age: 31.5 ± 6.2 years). Among them, 41.2% (161/391) self-reported using at least one antibiotic in the past year, with amoxicillin being the most common. Chlamydia prevalence was 8.2% (32/391), while no gonorrhea cases were detected. After adjusting for age, age at first debut, and engaging in high-risk sexual behaviors, prior use of any antibiotics was associated with a lower prevalence of chlamydia infection (OR: 0.39, 95% CI: 0.16, 0.95). Among patients who had used the recommended regimens for chlamydia infection (azithromycin, amoxicillin, and/or erythromycin), the prevalence was low (5.3%). It showed a non-significant trend toward reduced prevalence of current chlamydia infection (OR = 0.40, 95% CI: 0.14, 1.10).</p> Conclusions <p>Recent antibiotic use is prevalent among women attending gynecological clinics in China. Overall, prior antibiotic use showed a trend towards being negatively associated with the prevalence of chlamydia infection. Further investigations in larger studies are warranted.</p>