Streptococcal strains of bacteria belong to the major pathogen groups inducing those causing bovine mastitis. They not only negatively impact economic profit due to milk losses and therapy costs, but they are an important animal health and welfare issue as well. Thus, antimicrobial therapy is the main way of treatment for dairy cows infected with mastitis-causing microorganisms. However, selective pressure on bacteria caused by excessive prescribing and using of antibiotics in veterinary medicine, is one of the main causes of bacterial resistance to antibiotics. In addition to the possibility of using antibacterial alternatives, a reduction in the prevalence of antibiotic-resistant bacteria can be achieved by combining several substances (either pure alternatives or other substances with antibiotics) at significantly lower concentrations than the antibacterial dose alone. The aim of this study was to evaluate in vitro combinatory effect of gentamicin and zinc pyrithione against three bovine mastitis strains, especially Streptococcus agalactiae (CCM 6187, DSM 6784) and Streptococcus dysgalactiae (DSM 20662), by the standardized microdilution checkerboard method in 96-well microtitration plates. The minimum inhibitory concentrations (MICs), that are necessary to evaluate the fractional inhibitory concentrations (FICs), were determined as the lowest concentration limiting the growth of bacteria in wells compared to a positive control by ≥80%. The combinatory effect of gentamicin and zinc pyrithione was evaluated by values of FICs indices: synergy (FICs ≤0.5); indifference (FICs 0.5≤4); antagonism (FICs >4). Results showed significant synergistic effect against all tested streptococcal strains. The lowest FIC was found in Str. dysgalactiae (DSM 20662) (FIC 0.22). However, another tested strains Str. agalactiae (CCM 6187, DSM 6784) showed comparable values of FICs. Regarding the synergistic effect, values of FICs ranged from 0.22 to 0.39.