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This study aimed to investigate the chemical composition and the activity against Staphylococcus aureus (S. aureus) (ATCC 25923), Streptococcus pyogenes (S. pyogenes) (ATCC 19615), Escherichia coli (E. coli) (ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa) (ATCC 27853), Shigella flexneri (S. flexneri) (ATCC 12022), Salmonella typhimurium (S. typhimurium) (ATCC 14028), Haemophillus influenzae (H. influenza) type B (ATCC 10211) and two fungal strains: Candida albicans (C. albicans) (ATCC 10231) and Candida parapsilopsis (C. parapsilopsis) (ATCC 22019) of the extracts obtained from Melilotus officinalis (MO), Coronilla varia (CV); Ononis spinosa (OS) and Robinia pseudoacacia (RP) (Fabaceae), and to identify the chemical compounds responsible for the antimicrobial effect against the tested strains. The extracts were obtained by conventional hydroalcoholic extraction and analyzed in terms of total polyphenols using the spectrophotometric method and by liquid chromatography (LC). The results have shown that the highest polyphenols content was recorded in the RP sample (16.21 mg gallic acid equivalent GAE/g), followed by the CV (15.06 mg GAE/g), the OS (13.17 mg GAE/g), the lowest value being recorded for the MO sample (11.94 mg GAE/g). The antimicrobial testing of plant extracts was carried out using the microdilution method. The most sensitive strains identified were: E. coli, S. typhimurium, P. aeruginosa and S. pyogenes , while protocatechuic acid, gallic acid, caffeic acid, quercetin, rutin, and kaempferol were identified as the chemical compounds responsible for the antibacterial effect. The analysis of the correlation between the chemical composition and the antimicrobial effect proved a moderate (r > 0.5) positive correlation between rosmarinic acid and S. pyogenes (r = 0.526), rosmarinic acid and S. typhimurium (r = 0.568), quercetin and C. albicans (r = 0.553), quercetin and S. pyogenes (r = 0.605). Therefore, it suggested possible antimicrobial activity generated by these chemical components. The results recommend the Fabaceae plants as promising candidates for further research to develop novel natural antimicrobial drugs.