The molecular structures of N,N′-2,6-dimethylphenyl-o-phenylenediamine (1), N,N′-2,4,6-trimethylphenyl-o-phenylenediamine (2), N,N′-2,6-diisopropylphenyl-o-phenylenediamine 3a and 3b (dimorph of 3a) have been elucidated by X-ray diffraction as well as characterized by FT-IR, HRMS, 1 H, 13 C NMR and 2D experiments. The cytotoxic and antibacterial activities of the N,N′-diaryl-o-phenylenediamines (1–3) were tested against human tumor cell lines A431 and MOLT4 and Salmonella typhimurium and Staphylococcus aureus respectively. Compounds 1 and 2 showed higher biological activities than compound 3 in cell line A431 and against S. typhimurium. Tumor cell growing was inhibited with 1 and 2 1.0μg/mL and 0.5μg/mL, respectively. The minimal inhibitory concentration against S. typhimurium and S. aureus for both compounds was 0.35μg/mL. However, all compounds presented very similar activities in cell line MOLT4 (1–3: 0.5μg/mL) and S. aureus (1–3: 0.35μg/mL). A decrease of steric hindrance on phenylenediamine derivatives might influence on biological activity.