Novel thiazolotriazolopyrimidine derivatives (23–33) designed as potential adenosine A2A receptor (A2AR) antagonists were synthesized. Molecular docking studies revealed that all compounds (23–33) exhibited strong interaction with A2AR. The strong interaction of the compounds (23–33) with A2AR in silico was confirmed by their high binding affinity with human A2AR stably expressed in HEK293 cells using radioligand-binding assay. The compounds 24–26 demonstrated substantial binding affinity and selectivity for A2AR as compared to SCH58261, a standard A2AR antagonist. Decrease in A2AR-coupled release of endogenous cAMP in treated HEK293 cells demonstrated in vitro A2AR antagonist potential of the compounds 24–26. Attenuation in haloperidol-induced motor impairments (catalepsy and akinesia) in Swiss albino male mice pre-treated with compounds 24–26 further supports their role in the alleviation of PD symptoms.