5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (IBD), and prevention of inflammation-associated colorectal cancer (CRC). Its clinical effect is generally attributed to modulation of host inflammatory responses. However, the recent association of intestinal dysbiosis and selective enrichment of pathosymbiont Escherichia coli, including AIEC, in people and dogs with IBD and human CRC, raises the possibility that 5-ASA might also affect the enteric microflora. The aim of this study was to investigate the effect of 5-ASA on the growth and virulence of pathosymbiont E. coli associated with IBD and CRC, and its impact on host cell inflammatory responses. Our results show that 5-ASA inhibited E. coli growth in a dose-dependent manner and down-regulated the expression of bacterial virulence genes associated with IBD (fliC, fimH, ompC, yfgL, nlpL, lpfA, htrA, dsbA, fyuA, and chuA) and CRC (pks). 5-ASA inhibited E. coli motility, epithelial adherence and invasion, and IL-8 secretion. 5-ASA reduced E. coli survival in J774A.1 macrophages and TNF-α secretion by infected macrophages. In addition, 5-ASA reduced DNA damage in epithelial cells (Caco-2) induced by pks-positive E. coli. Our results reveal that 5-ASA impacts the growth and virulence of IBD- and CRC-associated E. coli, in addition to modulation of host inflammatory responses. These results suggest that 5-ASA may abrogate the proinflammatory and oncogenic effects of pathosymbiont E. coli, including AIEC, linked to IBD and CRC.