Microbial infection is one of the most significant causes of ear diseases, but microbial profiles are very diverse according to the diseases and change over time. The purpose of the study was to clarify differences and chronological changes in causative pathogens among infectious ear diseases over the last 20 years, and to identify antibiotic resistance. In total, 1191 isolates were included from patients diagnosed with chronic otitis media without cholesteatoma (COM), cholesteatomatous otitis media (Chole), middle ear effusion (MEE), including acute otitis media and otitis media with effusion, and external otitis (EO). Data were collected periodically for the years 1995, 2000, 2004, 2009, and 2013. Culture results and antibiotic resistance were assessed. The most common microorganism identified was S. aureus. The microbial profiles differed significant among the COM, Chole, and MEE groups (p < 0.001). In contrast, there was no distinct difference between COM and EO (p = 0.332). COM, Chole, and MEE also showed significant chronological changes in microbial profiles over time. The frequency of CNS increased markedly in COM and Chole (p = 0.029 and 0.028, respectively): however, S. pneumoniae infection decreased significantly in MEE (p = 0.016). Methicillin-resistant S. aureus (MRSA) demonstrated a constant trend (p = 0.564), whereas ciprofloxacin-resistant P. aeruginosa increased over time (p < 0.001). Microbial profiles have changed over a 20-year period. Increases in the frequency of coagulase-negative Staphylococcus (CNS) and bacterial resistance to ciprofloxacin, used widely in treating ear infections, are noteworthy.