Background: Calixarenes are widely used macromolecules in supramolecular chemistry synthesized by simple phenol formaldehyde condensation reaction in the presence of base. This article describes the synthesis, antibacterial, and antifungal properties of calix[4]arene appended with pyrrolidine at the upper rim (CAP3). Methods: Antimicrobial activity of CAP3 was determined by Kirby-Bauer well agar diffusion method in Mueller-Hinton agar (MHA) medium of growth against Escherichia coli (ATCC 8739), Staphylococcus aureus (ATCC 10231) and Streptococcus viridans (ATCC 12392), Candida albicans (ATCC 32333), Aspergillus niger (ATCC 16404), and Aspergillus flavus (ATCC 90906). Structure-activity relationship was also used to evaluate the active site of CAP3.