Background: Several Host defence peptides (HDPs) are low molecular weight (< 50 amino acids residues) peptides detected in several ethnomedicinal plants and have particularly gained research interest in recent times. Due to their wide range of bioactivity, occurrence, abundance and ability to induce very little resistance, they hold promising potentials in drug development. This study investigated the presence of bioactive peptides in the roots of Calliandra portoricensis (CPr) (Mimosaceae) and evaluated its antimicrobial activity against gram-negative and gram-positive bacteria.Methods: The crude peptide extract was obtained and pre-purified on pre-loaded tube of RP-C 18 solid phase cartridges (strata giga tube C18-E; 5 g, 20 mL, Phenomenex, Germany). Peptide enriched fraction was chemically analysed for arginine-rich/aromatic amino acid-rich peptides using a modified G-250 analytical stain and ninhydrin on thin layer chromatography (TLC) for a preliminary screening. Furthermore, MALDI TOF/TOF peptidomics was used to detect the presence and masses of the peptides. Extracts from CPr were used to test the ability to inhibit microbial growth using p-INT (Para-iodonitrotetrazolium violet) dye, with 0.1% gentamycin as positive control. The concentration that inhibits the growth of microorganisms by 50% (IC 50 ) were determined. Toxicity of the two extracts was accessed using freshly hatched nauplii of Artemia salina. Data analysis were evaluated using Microsoft excel and GraphPad Prism5.Results: Low molecular weight (LMW) peptides were detected in CPr using TLC and MALDI-TOF MS. Generally, the extracts exhibited good inhibition (70-95%) against the gram-negative and gram-positive bacteria, except MRSA6 typed strain. Enhanced activity was observed in the pre-purified peptide fraction than in the methanol crude, except on MRSA6. The greatest antimicrobial inhibition by pre-purified peptide fraction was against MRSA22 (IC 50 = 0.69 ± 0.33 μg/mL). The crude methanol extract (LC 50 = 5.13 μg/mL) was slightly more toxic than the peptide extract (LC 50 = 6.12 μg/mL).Conclusions: This is the first report on detection of bioactive LMW peptides in Mimosaceae family. These peptides appear to be rich in arginine and aromatic amino acids. The peptide extract, in its pre-purified form showed a lower Brine shrimp cytotoxicity and an enhanced antimicrobial activity against the tested gram-negative and grampositive bacteria.