The dataset is composed of three experimental groups (i.e. ctrl, sublethal dose, lethal dose, as defined by preliminary cytotoxicity screening), three biological replicates each. The goal is to shed light on the physiological impacts produced by 20 nm polystyrene nanoplastics, using a gilthead sea bream Sparus aurata cell line (SAF-1), in compliance with New Approach Methodologies strongly encouraged by latest supranational agencies.