Herbicide-resistant biotypes emerge due to the heavy reliance on the use of identical herbicides in weed management. Despite this, chemical weed control remains popular among growers because synthetic herbicides are cost-effective and easy to use. However, there is an urgent need for herbicide innovation to address the issue of resistance. This study aimed to explore new herbicide options by synthesizing metal oxide nanoparticles. Calcium oxide was produced and evaluated for its phytotoxic effects against three common and widespread weeds in Malaysia. The phytotoxicity of calcium oxide nanoparticles derived from cassava peels (CaO NPs-CP) and cassava leaves (CaO NPs-CL) was tested in both soil and soilless conditions using the seeds of Eleusine indica, Tridax procumbens, and Ageratum conyzoides. The results indicated that CaO NPs-CP exhibited stronger phytotoxic effects than CaO NPs-CL. Further investigation showed that CaO NPs-CP are effective inhibitors of germination and root growth, with their herbicidal activity being concentration-dependent. These findings suggest that CaO NPs-CP have the potential for further exploration in developing novel herbicide formulations.