Control of foodborne pathogens in fresh produce is crucial for food safety, and numerous Salmonella Typhimurium (ST) outbreaks have been reported already. The present study was done to assess effectiveness of cold oxygen plasma (COP) against biofilms of ST mixed with cultivable indigenous microorganisms (CIM). ST and CIM were grown at 15 °C as monocultures and mixed cultures for planktonic state, biofilm on stainless steel, and lettuce leaves. Thereafter, the samples were treated with COP and surviving populations were counted using plate counting methods. Biofilms and stomatal colonization were examined using field emission scanning electron microscopy (FESEM) and food quality was assessed after treatment. Mixed cultures of ST and CIM showed an antagonistic interaction on lettuce but not on SS or in planktonic state. Mixed cultures showed significantly (p < 0.05) greater resistance to COP compared to monoculture biofilms on lettuce but not on SS or planktonic state. Shift from smooth to rugose colony type was found for planktonic and for biofilms on SS but not on lettuce for ST. Mixed culture biofilms colonized stomata on the inside as demonstrated by FESEM. Although, lettuce quality was not affected by COP, this technology has to be optimized for further development of the successful inactivation of complex multispecies biofilm structures presented by real food environment.