Background: Pneumococcal disease is still considered a global problem. With the introduction of pneumococcal conjugate vaccines (PCVs) serotype epidemiology changed, but antimicrobial resistance persists constituting a serious problem. The current study aimed to determine the serotype distribution and the antimicrobial susceptibility of recent Streptococcus pneumoniae isolates, following implementation of the 13-valent conjugate vaccine (PCV13). Materials and Methods: From January 2017 to December 2022 we evaluated 116 nonduplicate S. pneumoniae isolates collected from adult patients (21 - 98 years) cared for in the University Hospital of Heraklion, Crete, Greece. Pneumococcal isolates were serotyped by the Quellung reaction, and antimicrobial susceptibility testing was performed using E-test. Multidrug resistance (MDR) was defined as non-susceptibility to at least one agent in ≥3 classes of antibiotics. Results: Among the 116 isolates, 31% were recognized as invasive pneumococcal strains, while 69% were noninvasive. The isolates tested belonged to 25 different serotypes. The most prevalent serotypes were 11A (10.3%), and 35B (10.3%), followed by 3 (9.5%), 15A (7.8%), 25F (6.9%), 19A (5.3%), 35F (5.3%), and others (44.6%).The coverage rates of PCV13 and the pneumococcal polysaccharide vaccine (PPSV23) were 26.7% and 57.8%, respectively. PCV13 and PPSV23 serotypes decreased between 2017 - 2019 and 2020 - 2022, with a parallel increase in the non-vaccine types. Resistance rates to erythromycin, clindamycin, trimethoprim/sulfamethoxazole, penicillin, levofloxacin, and ceftriaxone, were 40.5%, 21.6%, 13.8%, 12.1%, 3.4%, and 0%, respectively. All isolates were susceptible to vancomycin, linezolid, and daptomycin. MDR was observed among 36 (31%) S. pneumoniae isolates. Conclusion The increasing levels of resistance in S. pneumoniae in Crete, Greece, highlight the need for continuous surveillance of antimicrobial resistance and development of strategies for its reduction, including antimicrobial stewardship programs, increased pneumococcal vaccination, and development of next generation PCVs with a wider serotype coverage.