Microbial resistance to antibiotics affects the control of clinical infections and is a growing concern in global public health. One important mechanism whereby microorganisms acquire resistance is biofilm formation. This context has led to the investigation of new antimicrobial substances from plants popularly used in folk medicine. In this work we studied the antimicrobial and antibiofilm activity of Zinnia peruviana roots, ziniolide (major root metabolite) and aerial parts against Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The minimum inhibitory and minimum microbicidal concentration and inhibition of biofilm production was determined. All Z. peruviana extracts showed antimicrobial activity, but that corresponding to the roots was the most active one. The best inhibitory and microbicidal activity was detected against Gram-positive bacteria (0.039 to 0.078 mg ml-1). The acetonic extract from Z. peruviana leaves showed moderate activity against Gram-positive bacteria (0.625 mg ml-1). Acetonic extract of Z. peruviana flowers showed weak activity (1.25 to 5 mg ml-1). All the extracts tested showed inhibition of biofilm formation, as well as the ziniolide, however, roots and flowers extracts showed higher antibiofilm activity particularly against Staphylococcus, Listeria and Candida. The extracts tested may be a promising natural alternative for the control of microbial infections.