A series of 5-nitroisatin-3-thiosemicarbazones 2a–2l was synthesised and evaluated for selected biological activities. The brine shrimp lethality bioassay was carried out to study their in vitro cytotoxicity potential and besides, their antifungal, phytotoxic and urease inhibitory effects were also investigated. Only compound 2j proved to be active in the brine shrimp assay exhibiting LD50 value 1.16 9 10-3 M. Compounds 2a and 2d displayed moderate antifungal activity (50 and 40%, respectively) against M. canis. Similarly, compound 2l exhibited moderate activity (40%) against the fungal strain, A. flavus. In phytotoxicity assay, all the synthesised compounds including the reference point 2m showed weak to moderate (20–60%) activity at the highest tested concentrations (1,000 lg and 500 lg/ml, respectively). In urease inhibition assay, compounds 2a, 2i and 2k proved to be potent inhibitors demonstrating pronounced inhibition with IC50 values 0.440, 0.901 and 27.880 lM, respectively. These compounds may act as leads for further studies.