The concern against long-term health and environmental adverse effects of synthetic pesticides has encouraged the development of bio-pesticides. Eugenol, a major constituent of clove oil, has been proven as potential bio-pesticides. However, evaporation and photosensitive properties of Eugenol needs to be controlled. Nano-encapsulation is a promising method that can preserve eugenol from evaporating and photodegradation. This study aims to investigate the production of a controlled-release of eugenol in casein micelle as well as the effects of nano-encapsulation on Eugenol Containing Biopesticide (ECB) toxicity against Artemia salina sp. Brine Shrimp Lethality Test (BSLT) was implemented to investigate effect of nano-encapsulation on ECB and the Response Surface Methodology was used to optimize the formula to investigate the production of a controlled-release of eugenol. The optimum condition revealed loading capacity and encapsulation efficiency response for 64.67% and 79.64%, respectively. The average diameter of the obtained nanocapsule-eugenol (NCE) was 179.83 nm. Release study was performed at 40 °C that represent as pesticide applied in farm, revealed that casein micelle capsule could delayed the release of eugenol. A cytotoxicity assay showed that the NCE has 21 times more effective compared with eugenol only. It was found that nano encapsulated ECB was statistically more toxic than ECBsuspension (without nano encapsulation) with a confidence level of 95%. Lethal Concentration 50 (LC50) of nano-ECB was 0.264 μg/L while LC50 of ECB-suspension was 4.445 μg/L. The increase of toxic properties after nano-encapsulation by casein could be explained by the increase of eugenol stability. Thus nano-encapsulation can be proposed as a method for improving the bio-pesticide ability of eugenol.