The bisthiadiazolines 4a(a′-f′) and 4b(a′-f′) built around the various rigid chains have been synthesized in good yields by refluxing bisthiosemicarbazones 3a(a′-f′) and 3b(a′-f′) in acetic anhydride medium. The reaction of bisaldehydes 2a(a′-f′) and 2b(a′-f′) with thiosemicarbazide under alcoholic medium yielded 3a(a′-f′) and 3b(a′-f′) and the former were obtained from the reaction of 2/4-hydroxybenzaldehyde with suitable alkylating agent in the presence of anhydrous K2CO3/dry acetone and Bu4N+I− (PTC). The intermediates and final compounds have been characterized from the rigorous analysis of their IR, 1H-NMR, 13C-NMR, ESI-Mass and elemental analysis. The antibacterial and antifungal activities of the prepared compounds were also evaluated against the Klubsellia pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Bacillius subtilis and Aspergillius janus and Pencillium glabrum strains, respectively. The formation and antimicrobial behaviour of the bisthiadiazolines 4a(a′-f′) and 4b(a′-f′) are found to be independent of nature of the internal spacer unit.