Recently, our in vitro study on the effects of plasma-activated simulated seawater (PASW) demonstrated its potential as a novel, alternative disinfectant in oyster depuration. In this current study, PASW’s physicochemical and antimicrobial properties were identified and its effects on oyster quality were observed. Simulated seawater (SS) activated by plasma for 10 min (PASW10) increased in temperature, oxidation reduction potential, and electrical conductivity with a decrease in those parameters after 1:1 dilution with SS (PASW10 (1:1)). The pH decreased immediately after plasma activation and subsequently increased after dilution. Nitrate and nitrite concentrations were higher in PASW10 (1:1) compared to PASW10 (3:1). No free available chlorine was detected in undiluted PASW10. Processing using SS and PASW10 (1:1) depuration and refrigeration of E. coli-bioaccumulated oysters was observed. Significantly higher reductions in total coliform and E. coli were noted for PASW10 (1:1) treated oyster samples after 24 h compared to refrigerated samples. No significant differences in values for condition index or biting force of oyster meats were observed for the processed samples after 24 h. Overall, the novel and environment-friendly PASW could be used as an alternative antimicrobial disinfectant in oyster depuration systems because it does not negatively affect an oyster’s viability or quality.