Background Mosquitoes transmit serious human diseases, causing millions of deaths every year. Use of synthetic insecticides to control vector mosquitoes has caused physiological resistance and adverse environmental effects in addition to high operational cost. Insecticides of botanical origin have been reported as useful for control of mosquitoes. Azadirachta indica (Meliaceae) and its derived products have shown a variety of insecticidal properties. The present paper discusses the larvicidal activity of neem-based biopesticide for the control of mosquitoes. Methods Larvicidal efficacy of an emulsified concentrate of neem oil formulation (neem oil with polyoxyethylene ether, sorbitan dioleate and epichlorohydrin) developed by BMR & Company, Pune, India, was evaluated against late 3rd and early 4th instar larvae of different genera of mosquitoes. The larvae were exposed to different concentrations (0.5–5.0 ppm) of the formulation along with untreated control. Larvicidal activity of the formulation was also evaluated in field against Anopheles, Culex, and Aedes mosquitoes. The formulation was diluted with equal volumes of water and applied @ 140 mg a.i./m2 to different mosquito breeding sites with the help of pre calibrated knapsack sprayer. Larval density was determined at pre and post application of the formulation using a standard dipper. Results Median lethal concentration (LC50) of the formulation against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was found to be 1.6, 1.8 and 1.7 ppm respectively. LC50 values of the formulation stored at 26°C, 40°C and 45°C for 48 hours against Ae. aegypti were 1.7, 1.7, 1.8 ppm while LC90 values were 3.7, 3.7 and 3.8 ppm respectively. Further no significant difference in LC50 and LC90 values of the formulation was observed against Ae. aegypti during 18 months storage period at room temperature. An application of the formulation at the rate of 140 mg a.i./m2 in different breeding sites under natural field conditions provided 98.1% reduction of Anopheles larvae on day 1; thereafter 100% reduction was recorded up to week 1 and more than 80% reduction up to week 3, while percent reduction against Culex larvae was 95.5% on day 1, and thereafter 80% reduction was achieved up to week 3. The formulation also showed 95.1% and, 99.7% reduction of Aedes larvae on day 1 and day 2 respectively; thereafter 100% larval control was observed up to day 7. Conclusion The neem oil formulation was found effective in controlling mosquito larvae in different breeding sites under natural field conditions. As neem trees are widely distributed in India, their formulations may prove to be an effective and eco-friendly larvicide, which could be used as an alternative for malaria control.