The rising use of botanical biopesticides in integrated pest management demands rigorous evaluation of their ecotoxicological impacts on non-target aquatic organisms. This present study investigates the toxic effects of three plant-derived oils—neem oil (Azadirachta indica), pungam oil (Pongamia pinnata), and coconut oil (Cocos nucifera) on the Poecilia sphenops (Molly fish). Thus, commercial oils and fish are purchased from the Palayamkottai market and local aquariums from Tirunelveli, Tamil Nadu, India (latitude 8°43059 N and longitude77°4425 E). All the fish are maintained under the laboratory conditions. The acute toxicity (96 h) and subacute toxicity (21 days) assays were conducted at concentrations of different ppm (1, 5, and 10). And operculum movement monitoring and histopathological examination of gill and muscle tissues were carried out. The neem oil exhibited the highest toxicity value, with LC₅₀ = 6.52 and LC₉₀ = 12.84, followed by the Pungam Oil value of LC₅₀ = 4.18 and LC₉₀ = 8.92, while coconut oil proved negligible toxicity, with LC₅₀ > 100. The operculum movement decreased significantly in a concentration- and time-dependent manner, with maximum respiratory depression observed at 10 ppm in Pungam oil 50 beats/min compared to 77 from the control group on the 4th day. During the subacute exposure, there was 100% survival for neem and coconut oils, whereas Pungam oil at 10 ppm caused 20% mortality. This study providing the botanical oil biopesticide like, the neem, Pungam, and coconut oil. And their differential toxicity profiles, particularly pungam oil's potent effects on Poecilia sphenops, demand careful dosage regulation in Agro-aquatic ecosystems to safeguard non-target fish species.