Streptococcus pneumoniae susceptibility to 12 antimicrobial agents was assessed using isolates collected from patients with invasive and non-invasive infections in a Spanish medical center. Two hundred and thirty-six invasive and 478 non-invasive pneumococcal isolates obtained between 1998 and 2004 were tested. Penicillin non-susceptible isolates were more likely to exhibit resistance to cephalosporins, macrolides, chloramphenicol, and tetracycline when compared to penicillin-susceptible isolates. Penicillin resistance was present in 8.1% of the invasive and 18.6% of the non-invasive isolates. Overall, antimicrobial resistance was greater in non-invasive versus invasive isolates in adults. Serogroups included in the 7-valent and 23-valent formulation accounted for approximately 92.8 and 88.3% of the invasive isolates in children 2 years old or younger and the elderly, respectively. The proportion of isolates not susceptible to penicillin, erythromycin, and/or tetracycline decreased significantly over the surveillance period. Local epidemiological data assisted in the clinical determination of treatment protocols and effective prevention strategies.