The main objective of this work is to investigate the impact of oral administration of ampicillin on the ecological balance of enterococci in the intestinal microbiota of rats during a treatment and a post-treatment. The results have showed that the treated animals excreted significantly higher percentages of resistant enterococci compared to the control group (P ≤ 0.05) during the treatment and after the treatment. The most predominant species selected after the treatment began were Enterococcus faecium. The MICs for ampicillin for all isolates of E. faecium were 32 to 64 µg/mL, with the exception of two strains (TR1LBMB, TR5LBMB), were found to be highly resistant (MICs ≥ 128 µg/mL). Quantification of ampicillin in faeces by the RT-HPLC showed that the significant increase in the number of ampicillin-resistant enterococci was associated with the gradual accumulation of high levels of unabsorbed ampicillin in the faeces. Our results suggest that ampicillin treatment can now be understood as a side effect contributing to the increase in the number of resistant Enterococcus strains, particularly E. faecium strains, recognized as important nosocomial pathogens.