Citreamicin alpha (LL-E 19085-alpha) is an antibiotic that has been evaluated for its in vitro antibacterial activity against 429 clinical isolates of Gram-positive cocci using the agar dilution method. This study included 313 strains of staphylococci and 116 strains of streptococci. Its in vitro efficacy was compared with ampicillin, amoxicillin, cephalothin, erythromycin, and vancomycin. For staphylococci, Citreamicin alpha demonstrated MIC values ranging from 0.12 to 4.0 μg/ml, and for Streptococcus pyogenes, the MIC values ranged from 0.03 to 0.12 μg/ml. Conversely, enterococci presented relative resistance, requiring 2.0 μg/ml to inhibit 64% of 62 tested strains. The antibiotic's in vitro efficacy significantly surpassed that of ampicillin, amoxicillin, cephalothin, and erythromycin, while it was comparable to or slightly less effective than vancomycin.