Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P.hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C.parapsilosis and C.tropicalis. Fractions and chalcones were tested against C.albicans and S.aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S.aureus and B.subtilis with MIC values between 15.6 and 62.5μg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C.albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C.albicans and S.aureus biofilm cells at concentrations of 62.5 and 200μg/mL, respectively. Thus, P.hispidum may be a possible source of bioactive substances with antimicrobial properties.