In 2009, 1462 Escherichia coli isolates were collected in a systematic resistance monitoring approach from primary production, slaughterhouses and at retail and evaluated on the basis of epidemiological cut-off values. Besides resistance to antimicrobial classes that have been extensively used for a long time (e.g. sulphonamides and tetracyclines), resistance to (fluoro)quinolones and third-generation cephalosporins was observed. While in the poultry production chain the majority (60%) of isolates from laying hens was susceptible to all antimicrobials tested, most isolates from broilers, chicken meat and turkey meat showed resistance to at least one (85–93%) but frequently even to several antimicrobial classes (73–84%). In the cattle and pig production chain, the share of isolates showing resistance to at least one antimicrobial was lowest (16%) in dairy cows, whereas resistance to at least one antimicrobial ranged between 43% and 73% in veal calves, veal and pork. Resistance rates to ciprofloxacin and nalidixic acid in isolates from broilers were 41.1% and 43.1%, respectively. Likewise, high resistance rates to (fluoro)quinolones were observed in isolates from chicken meat and turkey meat. In contrast, ciprofloxacin resistance was less frequent in E. coli isolates from the cattle and pig production chain with highest rate in veal calves (13.3%). Highest resistance rates to cephalosporins were observed in broilers and chicken meat, with 5.9% and 6.2% of the isolates showing resistance. In dairy cattle and veal, no isolates with cephalosporin resistance were detected, whereas 3.3% of the isolates from veal calves showed resistance to ceftazidime. Resistance to (fluoro)quinolones and cephalosporins in E. coli isolates is of special concern because they are critically important antimicrobials in human antimicrobial therapy. The emergence of this resistance warrants increased monitoring. Together with continuous monitoring of antimicrobial usage, management strategies should be regularly assessed and adapted.