A series of 3,6-dialkyl-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazole (10) analogues were prepared through multistep synthesis and evaluated them for their antimicrobial and cytotoxic activities. Synthesis of target compounds was carried out using undecenoic acid as starting material, which is the renewable product of castor oil. The key step in the synthesis was formation of triazolo [3,4-b][1,3,4]thiadiazole using various free fatty acids in presence of POCl3. It was observed that the undecenyl based triazolothiadiazole with butyl (6a), hexyl (6b) and lauryl (6f) derivatives exhibited promising antimicrobial activity against the tested strains.Particularly, Compound 6a exhibited the most promising activity with MIC value 3.9 μg/mL against most of the tested strains. It also showed potent minimum bactericidal concentration activity with MIC value 7.8 μg/mL against the tested strains. Cytotoxicity data revealed that most of the tested compounds revealed cytotoxic activity, Compounds 6b, 6d, 6f, 6g, 6h and 6i against SKOV3, 6d, 6e, 6f, 6g, 6h, 6i and 6j against MCF-7 and 6c, 6d, 6e,6g, 6h, 6i and6j against B16–F10 cell lines exhibited significant activities with IC₅₀ values ranged between 13.67 and 18.62 μM. Interestingly, all the compounds were non toxic against Chinese hamster ovary cell (CHO-K1) normal cell