Synthetic carotenoid analogs, with increased utility for biological applications, are sparingly reported in the literature. Synthetic modification, which may increase the water solubility and/or water dispersibility of lipophilic carotenoids, allows their use in aqueous environments as potent antioxidants against potentially deleterious reactive oxygen species (ROS) that can be generated in vivo. Superoxide anion, produced by activated human neutrophils, can be a source of additional harmful ROS and nonradical species such as singlet oxygen in vivo. In the current study, direct scavenging of superoxide anion by a well-characterized C30 carotenoid phospholipid mixture was evaluated in a standard in vitro isolated human neutrophil assay by electron paramagnetic resonance (EPR) spectroscopy, employing the spin-trap DEPMPO. The carotenoid phospholipid was tested in aqueous formulation (aqueous dispersibility >60 mg/mL), in which supramolecular assembly takes place, as well as in ethanolic formulation as a monomeric solution of the carotenoid phospholipids. The carotenoid phospholipid (a highly unsaturated zwitterionic surfactant) was compared with a previously characterized rigid, long-chain, highly unsaturated dianionic bolaamphiphile, which contains an additional three conjugated double bonds in its extended conjugated system. As previously reported, direct scavenging by the carotenoid phospholipid derivatives in monomeric ethanolic formulation was superior at each tested concentration to aqueous, aggregated formulations of the compounds. Additionally, the percent inhibition of superoxide signal was related to the apparent or effective length of the conjugated chromophore, consistent with previous reports of radical inhibition and singlet oxygen quenching by polyene carotenoids of differing length.