The incidence of ampicillin (ABPC)-resistant Escherichia coli (E. coli) infection in very low-birthweight infants has been increasing. The rate of ABPC/sulbactam (ABPC/SBT)-resistant E. coli in this population, however, is currently unknown. We encountered two cases of severe infection due to resistant E. coli and retrospectively studied the prevalence of ABPC- and ABPC/SBT-resistant E. coli in regular surveillance cultures obtained from all neonatal intensive care unit (NICU) patients between 2000 and 2013. The overall prevalence of ABPC-resistant E. coli was 39% (47/120), accounting for 63% of cases (32/51) between 2007 and 2013, compared with 22% (15/69) between 2000 and 2006. The prevalence of ABPC/SBT resistance was 17% (20/120), which was similar in both periods (16%, 8/51 vs 17%, 12/69). According to these results, not only ABPC, but also ABPC/SBT-resistant E. coli must be considered in the NICU.