Background Recurrent subclinical mastitis (RScM) due to resistant bacteria has low clinical and bacteriological cure rates, often requiring the culling of cows. The sequential intra-mammary administration of enrofloxacin hydrochloride-dihydrate (enro-C) followed by ceftiofur HCl may be useful for treating these cases. Objectives This study assessed the bacteriological and clinical cure-efficacies of the sequentially intramammary administration of enro-C, followed by ceftiofur HCl to treat RScM in Holstein/Friesian cows. Methods This trial was conducted in a herd with a high prevalence of RScM, and 20 Holstein/Friesian cows were included: 45% suffering subclinical mastitis and 38.9% of the mammary quarters affected. Twenty-nine bacterial isolates in vitro resistant to enro-C were obtained (coagulase-negative Staphylococcus spp, 55.2%; Staphylococcus aureus , 27.6%; Escherichia coli , 6.9%; Streptococcus uberis , 6.9%; Corynebacterium bovis , 3.4%). Polymerase chain reaction-isolated the following genes linked to enro-C resistance: chromosomal ( gyrA ) and plasmid ( aac(6')-lb-cr ). The treatments were as follows: twice-daily intramammary infusions of enro-C (300 mg/10 mL) for 5 days. Cows clinically considered treatment failures were also treated with intramammary ceftiofur (125 mg/10 mL, twice daily for 5 days. The clinical and bacteriological cure rates were carried out when completing each treatment phase and at 14 and 21 days, aided by a California mastitis test, somatic cell count, and failure to identify the initially causative bacteria. Results Enro-C achieved 65% clinical and bacteriological cure rates, and 100% cure rates were obtained after the rescue treatment with ceftiofur HCl. Conclusions Outstanding clinical and bacteriological cure rates in cows affected by RScM were achieved with the consecutive intramammary infusions of enro-C, followed by ceftiofur HCl.