P2X receptors (P2XRs) are ligand-gated membrane ion channels. ATP binds to open these ion channels and results in membrane depolarization. Hyperactivities and overexpression of P2XRs are related to various pathophysiological conditions such as chronic pain, inflammatory diseases, rheumatoid arthritis, various neurological disorders, and cancer. Inhibition of P2XRs is a potential drug target that is an emerging therapeutic tool for disease management. In the present study, 17 new compounds were synthesized based on 1,3-benzodioxole-5-carboxylic acid and were investigated for P2XRs inhibition. Ca<sup>2+</sup> influx assay was performed on P2XRs expressed h-1321N1 astrocytoma cell lines. The synthesized compounds exhibited high potency and selectivity towards h-P2X4R and h-P2X7R. In silico studies were carried out that began with the development of a homology model for h-P2X7R with subsequent molecular docking studies of the most potent P2XRs antagonists. 9o (N-((2-bromo-4-isopropylphenyl)carbamothioyl)benzo[d] [1,3]dioxole-5-carboxamide) was found to have significant inhibitory potential and selectivity for h-P2X4R with an IC<sub>50</sub> ± SEM of 0.039 ± 0.07 μM. Whereas, 9q (N-(quinolin-8-ylcarbamothioyl)benzo[d] [1,3]dioxole-5-carboxamide) was selective and most potent antagonist for h-P2X7R with an IC<sub>50</sub> ± SEM of 0.018 ± 0.06 μM. Both antagonists, 9o and 9q, exhibited a non-competitive negative allosteric mode of antagonism.