As a first step towards developing genetic option to control pest populations of Gambusia holbrooki, this study investigated the efficacy of oestradiol (E2) to feminise the species. The oestrogen was administered orally via food to neonates (Experiment 1) or embryos through gravid females (Experiment 2) at dosage between 50 and 400 mg kg–1 of feed. Two control groups consisted of (C1) normal feed and (C2) feed mixed with 70% ethanol. In Experiment 1, 100% feminisation was observed at all E2 concentrations except at 400 mg kg–1 where no treated individuals survived. There was a significantly (P < 0.05) lower mean survival rate (MSR) of all E2-treated juveniles than of controls (C1: 71.73 ± 22.86%; C2: 70.02 ± 18.26%), with 50 mg kg–1 showing the best MSR (66.38 ± 12.34%). In Experiment 2, 100% feminisation was achieved at E2 concentrations of 200–400 mg kg–1 food, with the best MSR (59.33 ± 12.54%) at 200 mg kg–1 concentration, which was, however, significantly lower than in control groups (C1: 79.96 ± 20.33%; C2: 77.09 ± 10.32%). The reproductive output and gestation period of gravid females were not affected by E2 exposure. The outcomes provide a framework for reliable sex reversal (feminisation) in this fish, paving way for developing genetic strategies to manage and eradicate this pest fish.