Selection and spread of Extended Spectrum Beta-Lactamase (ESBL) -producing Enterobacteriaceae within animal production systems and potential spillover to humans is a major concern. Intramammary treatment of dairy cows with first-generation cephalosporins is a common practice and potentially selects for ESBL-producing Enterobacteriaceae, although it is unknown whether this really occurs in the bovine fecal environment. We aimed to study the potential effects of intramammary application of cephapirin (CP) and cefalonium (CL) to select for ESBL-producing Escherichia coli in the intestinal content of treated dairy cows and in manure slurry, using in vitro competition experiments with ESBL and non-ESBL E. coli isolates. No selection of ESBL-producing E. coli was observed at or below concentrations of 0.8 µg/ml and 4.0 µg/ml in bovine feces for CP and CL, respectively, and at or below 8.0 µg/ml and 4.0 µg/ml, respectively, in manure slurry. We calculated that the maximum concentration of CP and CL after intramammary treatment with commercial products will not exceed 0.29 µg/ml in feces and 0.03 µg/ml in manure slurry. Therefore, the results of this study did not find evidence supporting the selection of ESBL-producing E. coli in bovine feces or in manure slurry after intramammary use of commercial CP or CL-containing products.