Gram-positive anaerobic cocci (GPAC), a large group of anaerobic bacteria, are the members of the normal microbiota that colonizes the skin and mucosal surfaces of the human body. However, in case of a wound or when the host becomes immunocompromised, GPAC can cause invasive and most frequently mixed infections. GPAC are the second most frequently isolated bacteria in anaerobic infections. Although the studies are limited, GPAC have been reported to develop resistance to antimicrobial drugs. The resistance of the pathogens to the antimicrobials and improper therapy can cause poor clinical outcomes. Therefore, monitoring of the resistance trends of regional clinically important anaerobic bacteria periodically is recommended. In our study, we aimed to determine the antimicrobial susceptibility profiles of clinically important GPAC. A total of 100 non-duplicated pathogenic GPAC isolates were collected from Marmara University Hospital between 2013 and 2015. The isolates were identified by using conventional methods, "matrix-assisted laser desorption ionization-time of flight mass spectrometry system (MALDI-TOF MS)" (VITEK MS; v3.0, bioMerieux, France) and 16S rRNA gene sequencing. Antimicrobial susceptibility test was carried out by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The following antimicrobials were tested: penicillin, amoxicillin/ clavulanic acid (AMC), cefoxitin, meropenem, clindamycin, erythromycin, tetracycline, tigecycline, chloramphenicol, moxifloxacin and metronidazole. The minimum inhibitory concentration (MIC) results were interpreted according to the breakpoints described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoints recommended by CLSI for cefoxitin, tetracycline and moxifloxacin, and breakpoint recommended by Food and Drug Administration (FDA) for tigecycline were used since there were no EUCAST breakpoints for these antimicrobials. MIC50 and MIC90 values were determined for erythromycin since the breakpoint was not described by EUCAST, CLSI or FDA guidelines. The identification results showed that the strains (n= 100) consisted of five different GPAC genus; Parvomonas (40%), Finegoldia (34%), Peptoniphilus (14%), Peptostreptococcus (10%) and Anaerococcus (1%). All of the organisms were susceptible to meropenem, tigecycline and metronidazole. The isolates were highly susceptible to penicillin, AMC, cefoxitin, and chloramphenicol, since the resistance rates against these antimicrobials were 5% or less. The resistance rates against clindamycin, tetracycline and moxifloxacin were 14%, 31% and 24%, respectively. In total, 11% of the isolates were multidrug resistant. Metronidazole and tigecycline displayed high in vitro activity against GPAC and both are appropriate antimicrobials for the selection of empiric therapy. The effectiveness of meropenem was also found high, but it was observed that this antimicrobial would be more appropriate to use in the treatment of severe mixed infections accompanied by other microorganisms with the resistance potential. Detection of penicillin and AMC resistant isolates, which are frequently used in the treatment of GPAC infection, requires periodic monitoring of the antimicrobial susceptibility patterns of GPAC. The high rates of resistance against clindamycin, tetracycline and moksifloxacin indicated that these antimicrobials should not be used for empirical treatment of infections without prior antimicrobial susceptibility testing.