A randomized, controlled field trial was performed in The Netherlands to determine the therapeutic efficacy of parenteral penethamate hydriodide (Leocillin®) against naturally occurring, chronic, streptococcal mastitis during lactation. Quarter milk samples were collected from subclinical cases of Streptococcus uberis or Streptococcus dysgalactiae mastitis to determine the effect of treatment on bacteriological cure and somatic cell count (SCC) at quarter level. A quarter was considered to be cured when the bacterial species, isolated prior to treatment, was not isolated from the quarter milk samples taken on days 10 and 20 post-treatment (bacteriological cure), or when a quarter milk SCC (QMSCC) was <250000 cells/ml on days 10 and 20 post-treatment (SCC cure). Longitudinal data analysis was performed to determine the effect of antibiotic therapy on SCC and milk yield at cow level. Bacteriological cure occurred in 59% of 29 treated quarters, while no cure was observed in any of the 21 untreated control quarters. Treatment resulted in a significant decrease in SCC at cow and quarter level in comparison with untreated controls. There was no significant effect of treatment on milk production. Antibacterial treatment of subclinical streptococcal infections during lactation also prevented clinical mastitis. Furthermore, the treatment may contribute to reduction of bulk milk SCC and to prevention of pathogen spread in dairy herds.