Gram-negative bacteria (GNB) with multidrug resistance pose a serious threat to public health. They are environmental pathogens frequently isolated from raw milk and mastitis in dairy cattle. This study was to examine the in vitro antimicrobial activities against 225 isolates belonging to six important species of GNB from mastitic raw milk samples of dairy herds in the Republic of Korea: Acinetobacter baumannii (n = 17), Citrobacter freundii (n = 19), Enterobacter cloacae (n = 54), Klebsiella pneumoniae (n = 55), Pseudomonas aeruginosa (n = 45), and Serratia marcescens (n = 35). In general, amikacin, gentamicin, and piperacillin exhibited strong antimicrobial activities against all bacterial species tested, whereas rifampin, cephalothin, cefazolin, and ampicillin were ineffective against most of the bacterial species tested. Wide differences were observed in the patterns of resistance among the bacterial species; in particular, resistance to kanamycin, streptomycin, tetracycline, and chloramphenicol was highly variable among the strains belonging to different bacterial species. Almost half of the GNB isolates (45.3%, 102/225) were resistant to 5 or more of 12 antimicrobial agents tested: P. aeruginosa (86.6%, 39/45) showed the highest resistance rate, followed by S. marcescens (65.7%, 23/35). This study indicates that multiple antimicrobial resistances are prevalent among GNB isolates from mastitic milk samples of dairy cattle in the Republic of Korea.