Introduction Anonymous and free screening clinics (CDAG) were established in France in 1988 with the aim of supplementing the HIV prevention and control system. Over time, their scope of services was expanded to include screening for hepatitis C, and later for hepatitis B. This article presents the evolution of HIV and hepatitis C testing activity within the CDAGs during the period 2001–2015, and of hepatitis B testing during the period 2006–2015.Method Each year, the CDAGs submitted to Santé publique France an aggregated report of their screening activity for HIV (anti-HIV antibodies), HCV (anti-HCV antibodies), and HBV (HBsAg), including the number of tests performed and the number of positive tests by sex and age group. The analyses were conducted taking into account CDAGs that did not respond in a given year. Results The analyses performed on a constant number of CDAGs show an increase in HIV and HCV screening activity between the 2001–2005 and 2006–2010 periods, followed by a stabilization, as well as an increase in HBV testing activity between 2006–2010 and 2011–2015. Conversely, a decrease in HIV positivity rates is observed (from 4.8%, 95% CI: [4.6–5.0] in 2001–2005 to 3.6% [3.5–3.8] in 2006–2010 and to 3.4% [3.3–3.4] in 2011–2015) and for HCV (from 11.1% [10.8–11.4] in 2001–2005 to 7.5% [7.2–7.8] in 2006–2010), followed by stable rates over the most recent period. The HBV positivity rate decreased between 2006–2010 and 2011–2015, from 8.9% [8.6–9.2] to 7.9% [7.7–8.1]. For both infections, the positivity rate was higher among men than among women. The number of tests performed in 2015 by all CDAGs was estimated at 390,056, 95% CI: [351,452–428,661] for HIV, 200,132 [184,154–216,110] for HCV, and 236,005 [217,571–254,439] for HBV.Conclusion The significant increase in the number of HIV and HCV screening tests between the first two study periods and the concomitant decrease in positivity rates may reflect an expansion of screening efforts to a population less exposed to these two infections. The same may be true for HBV between the last two study periods. The data highlight the need to better characterize patients to adapt targeted screening strategies and to strengthen screening efforts in the general population.