Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs),due totheir anti-microbial properties, are one of the most commonly utilized. Despite this, the effects of sub-lethal concentrations of AgNPs, especially on gut biofilms, has been poorly investigated. To address these issues, we investigated the proteomic response of a mono-species Escherichia coli gut biofilm, used as in-vitro human gut model, to chronic and acute exposure of sub-lethal concentrations(1 µg/mL) of AgNPs. We used a new gel and label-free proteomic approach based on shotgun nanoflow scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) that, respect to the traditional proteomic investigation, allows a higher dynamic range of quantification of the whole proteome. To assess all different possible exposure scenarios, we compared the total proteome of four samples: untreated cells, cells treated with AgNPs for 24h (acute treatment), for 96h (chronic treatment), and cells grown in the presence of AgNPs for 96h and treated again for 24h (acute and chronic treatment). Proteomic profiling provided new insight into the mechanisms exerted by AgNPs highlighting the important role of the bacterial biofilm in protecting intestinal epithelial cells. Among the 1917 proteins identified, 216 were ANOVA significant and several pathways resulted altered including biofilm formation, bacterial adhesion, ROS stress response and glucose utilization.