Objectives: Enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) are among the most frequent microorganisms causing traveller's diarrhoea. The aim of this study was to investigate the evolution of antimicrobial resistance in EAEC and ETEC causing diarrhoea in patients who had travelled to different developing countries, comparing two periods of time, 1994-97 and 2001-04. Methods: Overall, 134 EAEC and 190 ETEC clinical isolates were studied. The MICs of ampicillin, chloramphenicol, nalidixic acid, tetracycline, trimethoprim/sulfamethoxazole, ciprofloxacin and amoxicillin/clavulanic acid were determined by the Etest method. Detection of mutations in the quinolone-resistance determining region of the gyrA and parC genes was performed by PCR and DNA sequencing. Results: When antimicrobial resistance in EAEC and ETEC isolates was compared between the two periods of time, a statistically significant increase in resistance (P < 0.01) was observed in EAEC for chloramphenicol and amoxicillin/clavulanic acid, whereas in ETEC it was for trimethoprim/sulfamethoxazole, nalidixic acid, ciprofloxacin and amoxicillin/clavulanic acid. Mutations in the gyrA gene were found in all nalidixic acid-resistant isolates, whereas mutation(s) in both gyrA and parC genes were found in the ciprofloxacin-resistant isolates. Conclusions: The high percentage of resistance to quinolones in ETEC and EAEC isolated from travellers to North Africa and India is a matter for concern. These agents should therefore be used with caution in patients with traveller's diarrhoea returning from these geographical areas.