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To examine rates of acute infectious endophthalmitis after pars plana vitrectomy (PPV) in eyes that received intraoperative subconjunctival antibiotics versus eyes that did not.A retrospective, nonrandomized, comparative case series of 18,886 consecutive cases of transconjunctival 23-, 25-, and 27-gauge PPV over a 5-year period was performed. The impact of prophylactic intraoperative subconjunctival antibiotics on the development of acute infectious postoperative endophthalmitis was examined.Of 18,886 cases of PPV, 14,068 (74.5%) received intraoperative subconjunctival antibiotics, whereas 4,818 (25.5%) did not. Sixteen cases (0.085%, 1/1,176) of post-PPV endophthalmitis were identified. The incidence of endophthalmitis in eyes that received subconjunctival antibiotics was 0.078% (11/14,068 cases, 1/1,282), whereas the incidence in those that did not receive subconjunctival antibiotics was 0.10% (5/4,818 cases, 1/1,000). No statistically significant difference was identified in the incidence of endophthalmitis between those that received subconjunctival antibiotics and those that did not (P = 0.598). Microbial culture was performed in 11 cases with 6 culture-positive cases (5/8 cases that received subconjunctival antibiotics and 1/3 cases that did not).Prophylactic subconjunctival antibiotics were not associated with a significantly reduced rate of post-PPV endophthalmitis. With consideration of emerging multidrug-resistant bacteria, routine prophylactic subconjunctival antibiotics may not be justified.