ABSTRACTNon-beta-hemolytic streptococci (NBHS), also referred to asviridansstreptococci, represent an underestimated cause of human invasive diseases. Their resistance to antibiotics, including beta-lactam agents, often complicate their therapeutic management. A prospective multicenter study was conducted by the French National Reference Center for Streptococci between March and April 2021 to describe the clinical and microbiological epidemiology of invasive infections due to NBHS, excluding pneumococcus. A total of 522 NBHS invasive cases were collected. Distribution among streptococcal groups was:Streptococcus anginosus(33%),Streptococcus mitis(28%),Streptococcus sanguinis(16%),Streptococcus bovis/equinus(15%),Streptococcus salivarius(8%), andStreptococcus mutans(<1%). Median age of infection was 68 years old (range <1 day to 100 years). Cases were more frequent in male patients (gender ratio M/F 2.1:1) and manifested mainly as bacteremia without focus (46%), intra-abdominal infections (18%) and endocarditis (11%). All isolates were susceptible to glycopeptides and displayed low-level inherent gentamicin resistance. All isolates of theS. bovis/equinus,S. anginosus, andS. mutansgroups were susceptible to beta-lactams. Conversely, nonsusceptibility to beta-lactams was found in 31%, 28%, and 52% ofS. mitis,S. salivarius, andS. sanguinisisolates, respectively. The screening for beta-lactam resistance using the recommended one unit benzylpenicillin disk screening failed to detect 21% of resistant isolates (21/99). Last, overall resistance rates to the alternative anti-streptococcal molecules clindamycin and moxifloxacin were 29% (149/522) and 1.6% (8/505), respectively.IMPORTANCENBHS are recognized as opportunistic pathogens particularly involved in infections of the elderly and immunocompromised patients. This study underlines their importance as common causes of severe and difficult-to-treat infections such as endocarditis. Although species of theS. anginosusandS. bovis/equinusgroups remain constantly susceptible to beta-lams, resistance in oral streptococci exceeds 30% and screening techniques are not fully reliable. Therefore, accurate species identification and antimicrobial susceptibility testing by MICs determination appears essential for the treatment of NBHS invasive infections, together with continued epidemiological surveillance.