This study aimed to synthesize and characterize cyclic tetrapeptides (CTPs) with potential pharmacological activities. The cyclic tetrapeptides were synthesized using N-methylmorpholine (NMM) as a base to facilitate the efficient cyclization of linear tetrapeptide segments. The percentage yields ranged from 77.5% to 81.6%, and the Rf values for CTP-1 and CTP-2 were 0.74 and 0.76, respectively. The synthesized CTPs were characterized by IR and 1H NMR spectroscopy. The disappearance of specific absorption bands and singlets corresponding to ester and tert-butyl groups confirmed successful cyclization. Antimicrobial activity was evaluated against Staphylococcus aureus and Escherichia coli by measuring the zones of inhibition (ZOI) at various concentrations. CTP-1 and CTP-2 demonstrated significant antimicrobial activity, with CTP-2 showing higher efficacy than Ciprofloxacin at 500 μg/mL. The COX-1 and COX-2 inhibitory assays indicated that both CTP-1 and CTP-2 exhibited significant inhibition at 250 μg/mL, suggesting strong anti-inflammatory properties. Additionally, the ABTS radical decolorization assay revealed concentration-dependent scavenging activity, with IC50 values comparable to quercetin and vitamin C. In conclusion, the synthesized cyclic tetrapeptides exhibited promising antimicrobial, anti-inflammatory, and antioxidant activities, highlighting their potential as therapeutic agents and warranting further pharmacological exploration.