The aim was to investigate the antimicrobial susceptibility of most frequently isolated streptococci from Czech dairy herds. A total of 3,719 quarter milk samples were collected and cultivated between January 2017 and June 2018 from cows with clinical or subclinical mastitis from 112 farms. Only one isolate of each species, collected from the same farm per six-month period, was included in the susceptibility testing. The susceptibilities of Streptococcus uberis (163 isolates) and S. dysgalactiae (25 isolates) to 10 antimicrobials (penicillin-PEN, amoxicillin/clavulanic acid-AMC, ceftiofur-EFT, clindamycin-CLI, gentamicin-GEN, streptomycin-STR, trimethoprim/sulfamethoxazole-SXT, enrofloxacin-ENR, tetracycline-TET, rifampicin-RIF) from 9 groups were determined by measuring their minimum inhibitory concentrations. The percentages of resistant S. uberis isolates to the antimicrobials were as follows: TET (63.2%), STR (52.1%), CLI (30.1%), and RIF (2.5%). Intermediate susceptibility was found to RIF (63.2%), PEN (35%), ENR (2.5%), EFT (1.8%), and AMC (1.2%). All the S. uberis isolates were susceptible to GEN and SXT (100%). However, only 6.7% of S. uberis isolates were susceptible to all tested antimicrobials, and 38.7% of isolates were multidrug resistant (≥ 3 groups of antimicrobials). All the S. dysgalactiae isolates were susceptible to PEN, AMC, EFT, GEN, SXT, and ENR (100%). Resistant S. dysgalactiae isolates were found to TET (60%), STR (28%), CLI (12%), and intermediate to TET (24%) and RIF (20%). Sixteen percent of S. dysgalactiae isolates were multidrug resistant. The relatively high occurrence of (multiple) resistance, relative to mastitis pathogens, highlights the importance of monitoring this condition in dairy herds.