Retinopathy of prematurity (ROP) is a leading cause of blindness in premature infants. The condition is associated with DHA deficiency. This study aimed to investigate the effect of DHA supplementation on the occurrence of ROP in infants receiving oral oil drops. It is part of the Joinville DHA study, a non-parallel-group cohort study conducted from March 2020 to January 2023 at a public maternity hospital in Brazil. Infants born before 33 weeks of gestational age or with a birth weight ≤ 1500 g were recruited. Among 155 infants, 81 did not receive and 74 received DHA supplementation until complete vascularisation of the peripheral retina. There was a higher incidence of infants with ROP in the unsupplemented group (58·6 %) compared with the DHA group (41·4 %), but this difference was NS ( = 0·22). Unadjusted logistic regression analysis showed that patent ductus arteriosus and neonatal corticosteroids were significantly ( < 0·05) associated with ROP in both groups. In the DHA group, surfactant use was also associated with ROP ( = 0·003). After adjusting for important covariates, patent ductus arteriosus and neonatal corticosteroids continued to be significant for infants in the unsupplemented group (OR = 3·99;  = 0·022 and OR = 5·64;  = 0·019, respectively). In the DHA group, only surfactant use continued to be associated with ROP (OR = 4·84;  = 0·015). In summary, DHA supplementation was not associated with ROP. Further studies are necessary to better understand the relationship between DHA supplementation, ROP and associated comorbidities.