One hundred and forty-four Escherichia coli strains isolated from 144 diarrhoeic lambs (5 to 21 days old) from 38 flocks in Spain were serotyped and investigated for production of enterotoxins (LT and STa), verotoxins (VT1 and VT2), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-haemolysin (Hly) and enterohaemolysin (EntHly), for necrotic and lethal activities and for antibiotic resistance. The strains belonged to 39 different serogroups; however, 58% were of one of 13 serogroups (O4, O6, O7, O8, O9, O11, O23, O26, O77, O80, O101, O103 and O161) and only four of them (O8, O9, O11 and O77) accounted for 34% of strains. In total 10 (7%) toxigenic strains were detected: two LT+, two VT1+ EntHly+, four VT1+ EntHly-, one CNF1+ Hly+ and one CNF2+. The highest percentages of antibiotic resistance were reached in the group of antibiotics (tetracycline, streptomycin, sulphadiazine, ampicillin, kanamycin, neomycin, chloramphenicol, trimethoprim and cotrimoxazole) that are most generally used by Spanish veterinary clinics. We conclude that E. coli strains isolated from diarrhoeic lambs are not generally toxigenic and belong to a large number of serogroups.