The frequency and antimicrobial susceptibility of mastitis caused by bacteria in cattle from a dairy farm in the Conache district, Trujillo, Peru, between September and December 2015 were determined. Milk samples were collected from 140 quarters of 35 cows that were checked for mastitis by direct observation in a cup with black background. For the microbiological analysis, the samples were cultured in blood agar and MacConkey agar and incubated at 37 °C for 24 h. To determine gram-positive bacteria tests of catalase, coagulase, mannitol fermentation and hemolysis were performed, and for gram-negative triple sugar iron agar (TSI), lysine-iron agar (LIA), Simmons citrate, urease, indole, methyl red, Voges Proskauer and glutamate broth were used. Antimicrobial susceptibility was determined by the Kirby-Bauer method with ampicillin, clindamycin, doxycycline, erythromycin, streptomycin, gentamicin, oxacillin and rifampicin disks. Thirty one cows with bacterial mastitis were detected, where 76% had gram-negative bacteria and 24% had gram-positive bacteria. The most frequent gram-negative bacteria were E. coli (28%) and Klebsiella sp. (24%), and the most frequent gram-positive bacteria was Staphylococcus aureus (16%). Pseudomonas aeruginosa was resistant to most antibiotics except erythromycin (intermediate susceptibility). E. coli presented intermediate susceptibility to erythromycin and resistance to oxacillin and rifampicin. Staphylococcus aureus presented intermediate susceptibility to clindamycin and erythromycin and resistance to ampicillin.Se determinó la frecuencia y susceptibilidad antimicrobiana de bacterias causantes de mastitis en bovinos de un establo del distrito de Conache, Trujillo, Perú, entre septiembre y diciembre de 2015. Se recolectaron muestras de leche de 140 cuartos individuales de 35 vacas que se les hizo el control de mastitis mediante observación directa y taza de fondo negro. Para el análisis microbiológico se sembraron las muestras en agar sangre y agar Mac Conkey y se incubaron a 37 °C por 24 h. Para determinar las bacterias gram positivas se realizaron pruebas de catalasa, coagulasa, fermentación de manitol y hemólisis; y para las gram negativas se realizaron pruebas de agar-hierro-triple azúcar (TSI), agar lisina-hierro (LIA), citrato de Simmons, ureasa, formación de indol, rojo metilo, Voges Proskauer y caldo glutamato. La susceptibilidad antimicrobiana se determinó mediante el método de Kirby-Bauer con discos de ampicilina, clindamicina, doxiciclina, eritromicina, estreptomicina, gentamicina, oxacilina y rifampicina. Se detectaron 31 vacas con mastitis bacteriana, donde el 76% presentó bacterias gram negativas y 24% gram positivas. Las bacterias gram negativas más frecuentes fueron E. coli (28%) y Klebsiella sp. (24%), y la bacteria gram positiva más frecuente fue Staphylococcus aureus (16%). Pseudomonas aeruginosa fue resistente a la mayoría de antibióticos, excepto a la eritromicina (susceptibilidad intermedia). E. coli presentó susceptibilidad intermedia a la eritromicina y resistencia a la oxacilina y rifampicina. Staphylococvcus aureus presentó susceptibilidad intermedia a la clindamicina y eritromicina y resistencia a la ampicilina.