Postoperative endophthalmitis is a rare, but serious complication of pars plana vitrectomy (PPV). Subconjunctival cefuroxime injection has been the traditional choice for post vitrectomy endophthalmitis prophylaxis. Its effectiveness and safety in this context are however poorly understood and cases of retinal toxicity have been reported. The traditional standard subconjunctival antibiotic prophylaxis has been superceded in cataract surgery by intracameral antibiotic prophylaxis. The primary aim of this three centre non-randomised retrospective database cohort study of 7,532 PPV procedures was to identify the rate of endophthalmitis in cohorts of patients treated with intracameral or subconjunctival cefuroxime. A secondary aim was to estimate the achieved intraocular antibiotic concentrations of cefuroxime in eyes with intracameral versus subconjunctival administration using mathematical modelling. The overall incidence of postoperative endophthalmitis was 0.07% (5/7532). There were no cases of endophthalmitis in eyes receiving intracameral cefuroxime alone or in combination with subconjunctival cefuroxime (0/5586). Patients receiving subconjunctival cefuroxime alone had a higher incidence of endophthalmitis (0.22%, 4/1835), and there was one case of endophthalmitis in eyes not receiving any perioperative antibiotics (0.9%, 1/111). No cases of cefuroxime toxicity were identified. With subconjunctival cefuroxime, in the presence of a sclerotomy leak, we estimated the vitreous drug concentration to be higher than that for intracameral cefuroxime and potentially toxic. Intracameral cefuroxime appears to be a safe and efficient choice for prophylaxis against endophthalmitis after PPV. Small eyes with intraocular tamponade seem to be at particular risk of drug toxicity if cefuroxime is administered via the subconjunctival route.