Given the worldwide use of intravitreal (IVT) injections, the authors of this paper set out to evaluate the rate of culture positivity of the needles used in routine IVT injections and compare this positivity between 27-gauge and 30-gauge needle tips. With an endophthalmitis risk of 0.02% the source of the organisms is assumed to be ocular surface and especially lid margin, meibomian orifices, and also oral and nasal bacterial flora. Currently no data exists comparing the contamination rates between 27 and 30-gauge needles used in IVT injection. Participants for the study were enrolled from consecutive patients who were scheduled to undergo IVT injections of either ranibizumab, bevacizumab or triamcinolone acetonide regardless of the indication. The injections were performed in the operating room by the same surgeon following the same protocol each time. Immediately after the injection, a sterile forceps was used to remove the needle from the syringe and drop it into the brain-heart infusion broth. A control group was also designed in order to eliminate field contamination. One hundred and twenty-six IVT injections procedures were performed, 62 were performed using 27-gauge needles, and 64 injections were performed using 30-gauge needles. Twenty-seven (21%) needles were culture positive. No cases of endophthalmitis were reported. The number of culture-positive 27-gauge needles was eight, representing 29.7% of all positive used needles and 13% of all 27-gauge injections. The number of culture-positive 30-gauge needles was 19, representing 70.3% of all positive used needles and 29% of all 30-gauge injections. Culture-positivity was different between 27- and 30-gauge groups (p=0.022). However, after Bonferroni’s correction was applied for multiple comparisons, this difference was non-significant (p>0.05). Of the control needles, 3.9% were culture positive. The used needle and its matching control needle were culture-positive with the same organism in two samples, which proves field contamination resulted in the positive culture for the paired needles. Coagulase negative Staphylococcus, found in 17 (53%) needles, was the most frequent organism. The results suggest that the contamination rate between 27- and 30-gauge needles is similar during IVT injection.