Background: Our aim was to determine whether the proportion of women testing positive for chlamydia had changed over time after adjusting for demographic, clinical, and sexual risk factors. Methods: Computerized records for all heterosexual women attending a large urban sexual health clinic for the first time between 2003 and 2007 and were tested for chlamydia, were included. Chlamydia positivity and 95% confidence intervals were calculated and logistic regression was used to assess any possible change in chlamydia positivity over time after adjusting for demographic, clinical, and sexual risk factors. National chlamydia surveillance and testing data and data of the use of antibiotics effective against chlamydia were analyzed and trends over time evaluated using linear regression. Results: There were 10,498 chlamydia tests conducted among female clients presenting to the clinic for their first time over the 5 years (2003-2007). Chlamydia positivity was 5.9% (95% CI: 5.5%-6.4%). Chlamydia positivity increased each year from 4.2% in 2003 to 6.7% in 2007 (P <0.01). After adjusting for other factors, chlamydia positivity increased on average 12% per year (OR = 1.12; 95% CI: 1.05-1.20). The average daily defined dose of antibiotics effective against chlamydia prescribed in Australia declined significantly between 1992 and 2002 (from 20.2 to 12.6 per 1000 population; P <0.01) at a time when chlamydia positivity across Australia was increasing (from 9.4% to 10.7%; P = 0.06). Conclusions: These data suggest that the true prevalence of chlamydia in Australia is rising. One biologically plausible explanation that requires further study is that fewer antibiotics are being prescribed that would inadvertently also treat chlamydia infection.