AbstractA series of 5‐alkylamino and 5‐alkylsulfanyl derivatives of 1‐aryl‐2‐alkyl‐4‐nitro‐1H‐imidazoles 12–21, 31, and 34 were synthesized by a simple method with the aim to develop novel HIV non‐nucleoside reverse transcriptase inhibitors (NNRTIs). All the new compounds were tested against HIV‐1 and HIV‐2 in MT‐4 cells. Compound 21, with an arylsulfanyl group at C(5) of the 4‐nitro‐1H‐imidazole backbone showed an EC50 value of 0.22 μg/ml against HIV‐1 with a therapeutic index of 13. This means that compound 21 was cytotoxic to MT‐4 cells at a CC50 value of 2.57 μg/ml; also compounds 8, 22–25, 28, and 29 were cytotoxic to MT‐4 cells within the 0.4–4 μg/ml concentration range. Compounds 8, and 12–21 were evaluated, as a rule, but found inactive at non‐toxic concentrations against hepatitis C virus, herpes simplex type 1 and 2, cytomegalovirus (CMV), varicella‐zoster virus (VZV), vaccinia virus, and vesicular stomatitis virus, and a number of other viruses. Yet, the therapeutic index of compounds 17 and 21 for CMV and VZV approached the tenfold cut‐off point. Compounds 8 and 21 exhibited some cytostatic activity against leukemia and melanoma cell lines.