In clinical treatment, the presence or absence of transudate coverage of exposed tumor tissue may have a significant impact on the therapeutic effect of cold atmospheric plasma. Here, the anticancer effects of a plasma jet on three‐dimensional multicellular tumor spheroids with or without liquid coverage were examined. It was found that cell viability decreased more rapidly without liquid coverage than with liquid coverage. Apoptotic cells and reactive oxygen species (ROS) were mainly distributed in the areas of the spheroids directly irradiated by the plasma jet without liquid coverage. However, with liquid coverage, they were distributed throughout the outermost layers of the whole spheroids. Importantly, liquid can help ROS penetrate into spheroids and prolong the anticancer effects of plasma.