The antimicrobial resistance and serotype distribution of Streptococcus pneumoniae (SP) among children attending day-care centres in south-eastern France were monitored from 1999 to 2008, before and after interventions promoting prudent antibiotic use initiated in 2000 and the availability of pneumococcal conjugate vaccine in 2003. Antibiotic susceptibility and serotypes of SP isolates were determined on nasopharyngeal samples of children aged 3-40 months attending day-care centres, from January to March 1999, 2002, 2004, 2006 and 2008. SP carriage fell from 54% to 45%, and SP with diminished susceptibility to penicillin (PDSP) fell from 34% to 19%. Antibiotic prescriptions dropped from 63% to 38% of children, but third-generation cephalosporins were increasingly prescribed. The overall antibiotic susceptibility increased. Over 90% of the children had received at least one vaccine dose in 2008. Vaccine serotypes 6B, 9V, 19F and 23F (76%) in 1999 were replaced by non-vaccine types (95%) in 2008, among which were 15 (20%), 19A (15%), 23A/B (10%) and 6A (9%). Serotypes 6A, 19A and 15 accounted for over 50% of PDSP strains in 2008 versus 6% in 1999. Children now mostly harbour non-vaccine types; however, PDSP isolates are mainly recruited among these. Vaccine-related benefits may be threatened by combined vaccine- and antibiotic-driven selective pressure.