Escherichia coli is one of the most ubiquitous pathogens causing several life-threatening diseases. Currently, antibiotics are failing fast while rates of drug-resistant bacteria are increasing worldwide with almost no new drugs coming to market and even last-resort antibiotics are becoming ineffective. Anti-virulence is a new approach targeting bacterial virulence and unlike classic antibiotics is aimed at disarming pathogens rather than killing them or inhibiting their growth. Inactivation of the bacterial arsenal of virulence factors will attenuate pathogens and render them susceptible to natural host defenses. The effect of the FDA-approved drug diclofenac sodium at a sub-inhibitory concentration (¼ MIC) on some Escherichia coli virulence factors was evaluated phenotypically and genotypically. Phenotypically, biofilm formation ability, proteases production and motility behavior were found to be significantly decreased under the effect of diclofenac sodium. Genotypically, qRT-PCR was used to evaluate the relative expression levels of papC, fimH, ompT_m, stcE and flic genes regulating virulence factors production. Importantly, the relative expression levels of all the tested genes showed significant downregulation under the effect of diclofenac sodium. The current research revealed that diclofenac sodium has a promising anti-virulence effect against resistant Escherichia coli. As a result, it is suggested that, in addition to or as an adjuvant to traditional antimicrobials, diclofenac sodium could be a possible solution to treat infections caused by resistant Escherichia coli.