Active vitreous seeds in eyes with retinoblastoma (Rb) adversely affects the treatment outcome. This study aimed to investigate the safety and efficacy of intravitreal melphalan chemotherapy (IViC) as a treatment for recurrent and refractory vitreous seeds in patients with Rb. We used a retrospective non-comparative study of patients with intraocular Rb who had vitreous seeds and were treated by IViC (20-30 μg of melphalan) using the safety-enhanced anti-reflux technique. Tumor response, ocular toxicity, demographics, clinical features, and survival were analyzed. In total, 27 eyes were treated with 108 injections for recurrent (16 eyes) or refractory (11 eyes) vitreous seeds after failed systemic chemotherapy. A total of 15 (56%) were males, and 20 (74%) had bilateral disease. At diagnosis, the majority ( = 21) of the injected eyes were group D, and = 6 were group C. Vitreous seeds showed complete regression in 21 (78%) eyes; 100% ( = 10) for eyes with focal seeds; 65% ( = 11/17 eyes) for eyes with diffuse seeds ( = 0.04); 7 (64%) eyes with refractory seeds; and 14 (87%) eyes with recurrent seeds showed complete response ( = 0.37). In total, 16 (59%) eyes developed side effects: retinal toxicity (48%), pupillary synechiae (15%), cataracts (30%), iris atrophy (7%), and retinal and optic atrophy (4%). Only one child was lost to follow-up whose family refused enucleation and none developed orbital tumor recurrence or distant metastasis. IViC with melphalan is effective (more for focal than diffuse seeding) and a relatively safe treatment modality for Rb that can improve the outcomes of eye salvage procedures. However, unexpected toxicity can occur even with the standard dose of 20-30 μg.