Although the iron-acquisition systems of Escherichia coli and Klebsiella pneumoniae are known to be important for pathogenicity, the interaction between the antimicrobial potential of lactoferrin (LF) and bacterial pathogenesis via iron uptake systems in bovine mastitis is still unknown. This study aimed to evaluate the effects of the LF concentration in milk and the bacterial iron-acquisition system on bacterial growth in unpasteurized raw milk from cows. Twenty-four strains of E. coli and 20 strains of K. pneumoniae, including mastitis-derived and environmentally derived strains, were used. The growth potential of these strains was tested by incubation with unpasteurized raw whole milk and cell-free skim milk from a total of 30 quarters. The LF concentration in milk and somatic cell counts (SCCs) were measured as host factors, whereas ferric citrate, siderophores, ferrous iron, and heme iron acquisition-related genes were detected as pathogen-related factors. For E. coli, strains with fecA grew better in milk, whereas host factors, including LF levels and the SCC in milk, did not affect bacterial growth in milk. In K. pneumoniae, the presence of fecA was also related to its ability to grow in milk. LF levels and SCCs in milk were significantly and negatively correlated with bacterial counts at 6 h in the milk growth experiment. These results suggested that the ferric-citrate-uptake systems of E. coli and K. pneumoniae may strongly contribute to their proliferation in mammary glands in dairy cows, whereas a high LF concentration in milk may successfully inhibit the growth of K. pneumoniae.