Introduction: In the last few decades, pyrazoline-based substances have emerged as potential antimicrobial and anticancer candidates. In concern with antimicrobial activity, this study aims to build a docking model to predict the structure of potential 2-pyrazoline derivatives. The cytotoxicity of some compounds was also evaluated to get insight into the structure–anticancer activity relationship of the 2-pyrazoline derivatives. Methods: Docking models were built on virtual FabH enzymes using FlexX platform with 2-pyrazoline derivatives served as test sets. Afterward, derivatives with high docking scores were chemically synthesized and evaluated for antibacterial activity using the agar dilution method. Furthermore, MTT assay was used to assess the cytotoxicity of these compounds. Results: The docking score and the in vitro minimum inhibitory concentration (MIC) value on Staphylococcus aureus (S. aureus) bacteria strongly correlate with an R-square value of 0.6751 (p < 0.0001). Four 2 pyrazoline derivatives were synthesized and evaluated for antimicrobial activity. Their MIC values on S. aureus range between 4 and 16 μg/mL, consistent with ones predicted by the docking model. Apropos cytotoxic properties, a series of 2-pyrazolines exhibit a moderate activity on HepG2, RD, and MDA-MB-231. The most active compound, HP10, has the IC50 values on these cell lines. which are 26.62 μM, 17.74 μM, 14.47 μM, respectively. Conclusion: Our research built a docking model on the virtual S. aureus FabH enzyme with high potential in predicting antibacterial activities of different 2-pyrazoline derivatives. Moreover, our cytotoxicity results provided data for further studies on the anticancer activity of these promising derivatives.