The aim of this study was to determine the characteristics and shifts in serotype distribution of pneumococcal isolates causing ocular infections in a region of northern Spain in two periods: 1999-2010 for episodes of conjunctivitis (n = 612) and 1980-2010 for uncommon and more severe non-conjunctival ocular infections (n = 36). All isolates were serotyped and non-typeable isolates were confirmed as unencapsulated by multiplex-PCR of the lytA, ply and cpsA genes. Genotyping was done by pulsed-field gel electrophoresis and multi-locus sequence typing. Most conjunctivitis cases occurred in children under 5 years old (89.5%), and more severe non-conjunctival ocular infections occurred in patients older than 25 years (86.1%). Unencapsulated isolates were detected in 213 conjunctivitis episodes (34.8%) and one non-conjunctival infection (2.8%). Rates of unencapsulated isolates were similar throughout the study. Among 399 conjunctival encapsulated isolates, the most prevalent were serotypes 19A (n = 53), 15B (n = 30), 6A (n = 27), 19F (n = 25), 23F (n = 21) and 6B (n = 17). The most prevalent serotypes in non-conjunctival infections were serotype 3 (n = 4), 23F (n = 4), 6B (n = 3) and 19A (n = 3). Conjunctivitis caused by serotypes included in the hepta-valent pneumococcal conjugate vaccine steadily decreased, accounting for 34.9% (22/63) in 1999-2001, 19.7% (23/117) in 2002-04, 13.6% (33/242) in 2005-07 and 3.2% (6/190) in 2008-10. Among the 213 unencapsulated isolates, 31 different pulsed-field gel electrophoresis patterns were identified. The main clonal complexes (CC) were CC941 (ST941, ST942), CC448 (ST448) and CC344 (ST344, ST3097). CC941 was the predominant CC in 1999-2001, 2002-04 and 2005-07, being replaced by CC448 in 2008-10. The multidrug-resistant CC344 was present throughout the study.