Background: Currently, the rise of antimicrobial resistance (AMR) has led to reduced therapeutic efficacy in severe infections such as osteomyelitis. Furthermore, broad spectrum synthetic antibiotics disrupt microbiome balance and weaken the host immune system. Therefore, given the need to investigate natural biologically active supplements that inhibit pathogens without adversely affecting the viability of beneficial microorganisms supported by experimental and clinical studies this study was grounded in determining the effects of the Osteo Calcium-5 supplement on pathogenic and beneficial bacteria Aim: To investigate the in vitro effects of the biologically active supplement Osteo Calcium-5 on pathogenic and beneficial bacteria. Objectives: To determine the antibacterial activity of the biologically active supplement Osteo Calcium-5 by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and the diameter of the inhibition zone. To compare the effects of the Osteo Calcium-5 supplement on the growth of pathogenic and beneficial bacteria, and to evaluate the levels of selective activity and resistance. Materials and Methods: The study was performed using a quantitative research approach with an experimental research design, utilizing two standard pathogenic bacterial strains Staphylococcus aureus ATCC 12600 and methicillin-resistant Staphylococcus aureus (MRSA) ATCC 700699 along with two standard beneficial bacterial strains, Lactobacillus paracasei and Streptococcus thermophilus. The Osteo Calcium-5 supplement was extracted using a solution of 70% ethanol and 0.1% HCl. To determine its antibacterial efficacy, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed in triplicate across a concentration range of 0.78% to 12.5% using the broth microdilution method, and evaluated via the agar well diffusion method based on the inhibition zone diameter. Results: It was established that the extract of the biologically active supplement Osteo Calcium-5 exerts selective effects on the growth of pathogenic versus beneficial bacteria. Specifically, for the pathogenic MRSA strain, the minimum inhibitory concentration (MIC) was 0.78% and the minimum bactericidal concentration (MBC) was 1.56%, whereas for the S.aureus strain, both MIC and MBC values were 1.56%, demonstrating high antibacterial activity with complete inhibition of colony growth at this concentration. In contrast, L.paracasei and S.thermophilus strains grew normally at low concentrations of the supplement, with MIC values ranging from 6.25% to 12.5%, proving to be several fold more resistant than the pathogenic strains. Furthermore, the agar diffusion method showed active inhibition of S.aureus (16.50±0.29mm) and MRSA (15.00-0.58mm) bacterial growth through the formation of inhibition zones, while exhibiting no adverse effects on the growth of the beneficial bacterial strains L.paracasei and S.thermophilus. Conclusions The MIC of the Osteo Calcium-5 supplement was determined to be 0.78% for MRSA and 1.56% for S.aureus, with the MBC for both strains established at 1.56%. Through the agar well method, the supplement actively inhibited the growth of S.aureus (16.50±0.29mm) and MRSA (15.00±0.58mm) by forming inhibition zones, effectively suppressing osteomyelitis inducing pathogens at low concentrations via mechanisms including bacterial cell wall and membrane disruption and virulence neutralization. For the beneficial bacterial strains L.paracasei and S.thermophilus, the MIC values of the supplement extract ranged from 6.25% to 12.5%, allowing normal growth at low concentrations without forming inhibition zones. This indicates a significantly higher resistance to the supplement compared to pathogens, proving its unique capability to selectively inhibit infections without disrupting the balance of the human normal microbiome.