Purpose To identify the microbial aetiology of infectious endophthalmitis and to determine the in vitro antibacterial susceptibilities of bacterial isolates. Methods A retrospective analysis was carried out of all patients presenting between January 1997 and December 2006 with clinically diagnosed infectious endophthalmitis who underwent microbiological evaluation. Intraocular specimens (aqueous and vitreous fluids) were collected from all cases of clinically suspected infectious endophthalmitis. In addition to intraocular aspirates, blood specimens from endogenous endophthalmitis, and corneal and scleral scrapes from relevant cases were also collected. The collected intraocular specimens, blood specimens, and corneal and scleral scrapes were subjected to microbiological evaluation. Results Samples from 955 patients with endophthalmitis underwent microbiological analysis, of which 424 (44.4%) were found to be culture positive. Of 424, 364 (85.8%) had bacterial growth and the remaining 60 (14.2%) had fungal growth. Among post-surgical endophthalmitis, Gram-negative bacilli (75%) were found to be the predominant cause for developing fulminant onset, Staphylococcus spp. (68.6%) for acute, and Streptococcus spp. (75%) for chronic onset of infections, whereas in post-traumatic endophthalmitis, Gramnegative bacilli (65.2%) were found to be the predominant cause for fulminant onset, Gram-positive bacillus (28.4%) for acute onset, and fungi (52.3%) for chronic onset of infections. Endophthalmitis associated with microbial keratitis was mainly caused by filamentous fungi (37.2%) and Gram-negative bacilli (37.2%). Overall, gatifloxacin (97.7%) showed highest activity against bacterial isolates followed by ciprofloxacin (95.9%) and ofloxacin (95.1%). Conclusion Gram-negative bacilli cause predominantly fulminant onset, Staphylococci and Gram-positive bacilli acute, and Streptococci, Nocardia, and fungi chronic endophthalmitis. Gatifloxacin demonstrated greatest efficacy against these bacterial isolates.