Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.