An efficient domino protocol has been developed for the synthesis of new pyrimidine scaffolds, through a one-pot four-component cascade transformation via [Bmim]HSO4 ionic liquid mediated reaction, using an equimolar mixture of thiochroman-4-one, benzaldehyde, thiourea and 3-bromo-1-phenylpropan-1-one leading to the formation of a double electrophilic pyrimidine-2(5H)-thione intermediate. The intermediate regioselectively undergoes cyclization through intramolecular NH bond activation followed by CS bond formation leading to highly functionalized thiazolo[3,2-a]thiochromeno[4,3-d]pyrimidines. The ionic liquid operates efficiently under mild conditions. The recyclability and scope for recovery of the ionic liquid makes this protocol environmentally benign. Further, the compounds 5d, 5g and 5k showed promising antimicrobial activity against the tested Gram-positive bacterial strains. Among them, the compound 5d was identified as a lead molecule exhibiting promising anti-biofilm activity towards Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 121, Staphylococcus aureus MLS16 MTCC 2940 and Micrococcus luteus MTCC 2470 with IC50 values of 2.1, 1.9, 2.4 and 5.3μg/mL, respectively. Further, the compound 5d showed increased levels of intracellular ROS accumulation in Staphylococcus aureus MTCC 96 suggesting that oxidative stress resulted in bacterial cell lysis and death.