Background Retinoblastoma is a rare intraocular malignancy in children. Current treatments have many adverse effects. New therapeutic approaches like intravitreal injections of chemotherapies are currently being developed but their toxicities need to be evaluated on animal models. This study compares the efficacy and toxicity of intravitreal melphalan, topotecan and carboplatin, alone or in combination (sequential administration), in the LHBetaTag retinoblastoma mice. Methods Mice were divided into nine groups: control, carboplatin 1.5 and 4 μg, melphalan 0.1 and 1 μg, topotecan 0.1 and 1 μg, carboplatin 4 μg/topotecan 0.1 μg and melphalan 1 μg/topotecan 0.1 μg. The follow‐up was performed using fundus imaging and optical coherence tomography combined with histopathological analysis. Absence of tumour and presence of calcified tumours were the criteria for therapeutic response assessment. Ocular complications were assessed after four weekly injections. Retinal toxicity was defined by the decrease of retinal thickness and of the number of retinal layers. Results Topotecan was inactive on retinal tumours. Melphalan (1 μg) led to a complete tumour control in 91.7% of eyes. Carboplatin strongly decreased the tumour burden (85.7‐93.8% of eyes without retinal tumour). The intravitreal injection itself led to ocular complications (25% of media opacities and 45.7% of retinal detachment). Only melphalan at 1 μg showed a strong retinal toxicity. The two combinations showed a good efficacy in reducing the number of eyes with retinal tumours with a reduced retinal toxicity. Conclusions This preclinical study suggests that intravitreal injection of carboplatin has a low toxicity and could be evaluated in clinical practice to treat patients suffering from retinoblastoma.