A series of mesoionic pyrimidinium betaines were synthesized and tested in vitro for their antioxidant activity using three tests: 2,2-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging, Ferric Reducing Antioxidant Power (FRAP) and bleaching of β-carotene. The results indicated that pyrimidinium betaines possess antioxidant properties with three methods. In the DPPH assay, the monocyclic pyrimidinium betaine showed the highest activity with an IC 50 value of 300 µg/mL. The pyrimidinium betaines also revealed a reducing power lower than that of the control antioxidants, and the monocyclic pyrimidinium betaine was the most active one. In the β-carotene bleaching test, the oxidation of linoleic acid was inhibited by all the betaines. The bicyclic one showed the greatest inhibitory activity of the oxidation of linoleic acid with a relative antioxidant activity (RAA) of 90%, an activity higher than that of the reference antioxidant α-tocopherol (41%). It was found that the antioxidant efficacy increased with increasing concentration of betaine and depended on the nature of the substituent on the pyrimidine ring.