Abstract Background Animals are a reservoir for ESBL/pAmpC-producing Escherichia coli/Klebsiella pneumoniae (ESBL-E/K). We investigated the association between occupational contact with different types of animals and the prevalence of ESBL-E/K carriage among veterinary healthcare workers, assessed molecular characteristics of ESBL-E/K, and followed-up on the ESBL-E/K carriage status of participants and their household members. Methods Participants completed a questionnaire about their contact with animals at work and at home, health status, travel behaviour and hygiene, and sent in a faecal sample which was tested for the presence of ESBL-E/K. Resistance genes were typed using PCR and sequencing . ESBL-E/K positive participants and their household members were followed up after 6 months. Risk factors were analysed using multivariable logistic regression methods. Results The prevalence of ESBL-E/K carriage was 9.8% (47/482; 95%CI 7.4–12.7). The most frequently occurring ESBL genes were bla CTX-M-15 , bla CTX-M-14 and bla DHA-1 . The predominant sequence type was ST131. None of the occupation related factors, such as contact with specific animal species, were significantly associated with ESBL-E/K carriage, whereas travel to Africa, Asia or Latin America in the past 6 months (OR 4.4), and stomach/bowel complaints in the past 4 weeks (OR 2.2) were. Sixteen of 33 initially ESBL-E/K positive participants (48.5%) tested positive again 6 months later, in 14 persons the same ESBL gene and E. coli ST was found. Four of 23 (17.4%) household members carried ESBL-E/K, in three persons this was the same ESBL gene and E. coli ST as in the veterinary healthcare worker. Conclusions Despite the absence of specific occupation related risk factors, ESBL-E/K carriage in veterinary healthcare workers was high compared to the prevalence in the general Dutch population (5%). This indicates that occupational contact with animals is a potential source of ESBL-E/K for the population at large.