N’-[(E)-(4-Chloro-3-nitrophenyl)methylidene]pyridine-4-carbohydrazide (Hyd) was synthesized via condensation of 4-chloro-3-nitro-benzaldehyde and isonicotinohydrazide (INH) in ethanol in the presence of a few drops of acetic acid. This synthesized compound was characterized by a number of spectroscopic methods including UV, IR, NMR (1H, 13C), and mass spectrometry, and by elemental analysis. Molecular structure properties of Hyd and INH were investigated using density functional theory (DFT) via B3LYP/6-31G (d,p) method, whereas the antioxidant effect was evaluated by superoxide anion scavenging assays. Results obtained from this investigation indicate high antioxidant effect of the Hyd with an IC50 value of 32.37 ± 0.45 μg/mL, which is comparable with that of ascorbic acid (AA). In addition, toxicity of the synthesized compound was evaluated by in vitro and in vivo bioassays using microorganisms and animals by molecular modeling using P values, and by predictions using Pred-Skin software program. Results show no toxic effect against bacteria and fungi at a high dose of 2000 μg/mL, no toxic effect against mice at a high dose of 1000 mg/kg, and low toxic effect towards the human skin. Biodegradability predictions of Hyd were carried out using online computer software programs (UM-BBD) and by in vitro assay using the degradation in mineral media. Results demonstrate biodegradation-ability of Hyd by simple microorganisms.