The multiple parallel synthesis of a series of N, S-bis-alkylated thiopyrazolo [3, 4-d] pyrimidines, based on sequential S-then N-alkylation, is reported. These compounds showed significant anti-mycobacterial activity (MICs down to⩽ 2μg/ml) and their potential as significant drug-like leads is substantiated through cytotoxicity evaluation and in silico profiling.