Ethanol extract and fractions from aerial parts of Eclipta alba (L.) Hassk (Asteraceae) were screened for the antibacterial and antifungal activities against different species of human pathogenic bacterial ATCC, antibiotic-resistant clinical isolates and strains of the dermatophyte Trichophyton rubrum (wild and mutant for TruMDR2 gene) using a microdilution method. Demethylwedelolactone/wedelolactone (DWL/WL) and only wedelolactone (WL), both in a high homogeneity degree, were efficient to inhibit the ATCC strains of Staphylococus aureus (Minimal Inhibitory Concentration MIC = 75 μg/mL), Staphylococcus epidemidis (MIC = 125 μg/mL) and Escherichia coli (MIC = 125 μg/mL) as well as antibiotic-resistant clinical isolates of Enterococcus spp (MIC = 250 μg/mL) and S. aureus (MIC = 125 μg/mL). Ethanol extract was more effective than the purified fractions against Trichophyton rubrum strains (MIC = 125 μg/mL), suggesting that anti-fungal activity is not only related to demethylwedelolactone and wedelolactone, but also to a synergistic action between these coumestans and other compounds found in that extract. Thus, this work suggests that E. alba possesses a significant antimicrobial activity, including that against multi-drug resistant microorganisms, which could be of relevance for the treatment of infectious diseases.