An 8-year-old male Rhodesian Ridgeback was presented with fever and severe thrombocytopenia. Clinical and laboratory examination, echocardiography, blood culture, and pathohistology revealed evidence of infective endocarditis, ischemic renal infarcts, and septic encephalitis. Treatment was started immediately but the dog's condition worsened, and the dog had to be euthanized. The causative strain was detected by blood culture and MALDI-TOF MS and analyzed using whole-genome sequencing and multilocus sequence typing. Antibiotic susceptibility testing did not detect any resistance. The affected heart valve was analyzed using FISH imaging, which showed a streptococcal biofilm on the heart valve. Bacteria in biofilms are recalcitrant to antibiotic treatment. Early diagnosis could be beneficial to treatment outcome. Treatment of endocarditis could be improved by researching the optimal dosage of antibiotics in conjunction with the use of biofilm-active drugs.