Food-producing animals, including dairy cattle, have been described as potential reservoirs of antimicrobial resistance. However, data remain scarce on the antimicrobial use and the selection of resistant bacteria. We investigated the association between antimicrobial use and resistance of mastitis pathogens, using data from 134 dairy farms in Chiba Prefecture, one of the major dairy production prefectures in Japan. Milk samples were collected from cows with mastitis in 2016. We recorded antimicrobial use and any isolation of methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamase (ESBL)-producing coliforms. The number of treatment days (TDs) was used as an indicator of the antimicrobial use on each farm. Farms in which either MRS or ESBL-producing coliforms were isolated from at least one mastitic milk sample were defined as an AMR positive farm. Farms from which neither MRS nor ESBL-producing coliforms were isolated were defined as AMR negative farms. The results revealed that the AMR positive farms had a significantly higher number of TDs (median value of 16.49) compared to AMR negative farms (median value of 14.02), suggesting that dairy cattle might be a source for the transmission of resistance genes and/or resistant bacteria between cattle and/or to humans.