Pesticides are widely used to eradicate insects, weed species, and fungi in agriculture. The half-lives of some pesticides are relatively long and may have the dire potential to induce adverse effects when released into the soil, terrestrial and aquatic systems. To assess the potential adverse effects of pesticide pollution in the aquatic environment, zebrafish (Danio rerio) and Daphnia magna are two excellent animal models because of their transparent bodies, relatively short development processes, and well-established genetic information. Moreover, they are also suitable for performing high-throughput toxicity assays. In this study, we used both zebrafish larvae and water flea daphnia neonates as a model system to explore and compare the potential toxicity by monitoring locomotor activity. Tested animals were exposed to 12 various types of pesticides (three fungicides and 9 insecticides) for 24 h and their corresponding locomotor activities, in terms of distance traveled, burst movement, and rotation were quantified. By adapting principal component analysis (PCA) and hierarchical clustering analysis, we were able to minimize data complexity and compare pesticide toxicity based on locomotor activity for zebrafish and daphnia. Results showed distinct locomotor activity alteration patterns between zebrafish and daphnia towards pesticide exposure. The majority of pesticides tested in this study induced locomotor hypo-activity in daphnia neonates but triggered locomotor hyper-activity in zebrafish larvae. According to our PCA and clustering results, the toxicity for 12 pesticides was grouped into two major groups based on all locomotor activity endpoints collected from both zebrafish and daphnia. In conclusion, all pesticides resulted in swimming alterations in both animal models by either producing hypo-activity, hyperactivity, or other changes in swimming patterns. In addition, zebrafish and daphnia displayed distinct sensitivity and response against different pesticides, and the combinational analysis approach by using a phenomic approach to combine data collected from zebrafish and daphnia provided better resolution for toxicological assessment.