In this study, we investigated the association between natural subclinical intramammary infection (IMI) caused by Streptococcus agalactiae and Prototheca spp. and milk differential cell counts assessed by cytofluorimetric analysis in Holstein cows. After an initial bacteriological screening on 188 animals and a second assessment carried out 2 wk later aimed at confirming the bacteriological status, we collected milk samples from 47 animals and performed (1) milk composition analyses; (2) somatic cell counts and differential somatic cell counts (DSCC); and (3) cytofluorimetric analyses. Before statistical analyses, animals with co-infections were filtered out. Bacteriological status (negative, positive for Strep. agalactiae, or positive for Prototheca spp.) significantly affected the investigated traits. Compared with culture-negative samples, those that were positive for Strep. agalactiae and Prototheca spp. had higher SCS (+61% and +49%, respectively), DSCC (+4% and +19%, respectively), log polymorphonuclear neutrophil (PMN)-lymphocyte (LYM) counts (+59% and +71%, respectively), and log macrophage (MAC) counts (+63% and +72%, respectively). The individual leukocyte populations determined by cytofluorimetric analysis confirmed that mastitis infection increased the proportion of PMN in the milk samples compared with culture-negative samples, particularly when caused by Strep. agalactiae (+51%). In the case of MAC, the 2 pathogens behaved in opposite ways: Strep. agalactiae increased MAC by 41%, whereas Prototheca decreased MAC by 25%. Prototheca infection strongly increased the proportion of total T lymphocytes (TL; +87%) and T-helper lymphocytes (+83%). Accordingly, the (PMN+MAC):TL ratio increased with Strep. agalactiae infection (+95%) and decreased with Prototheca infection (-43%) compared with culture-negative samples. These results suggest the prevalence of an adaptive immune response and chronic inflammation in Prototheca infection, and an innate immune response to Strep. agalactiae. This knowledge might provide an important contribution to the development of novel and effective diagnostics and therapeutics.