The World Health Organization (WHO) has prioritized developing new drugs against specific bacteria and fungi, such as Enterobacteriaceae and Candida spp. While Pfaffia paniculata is com- monly called the “cure-everything”, its scientifically proven benefits are limited to anti-inflammatory and antioxidant actions. Therefore, this study aims to determine the spectrum of antimicrobial activity of Pfaffia paniculata and assess its cytotoxicity. Thus, broth microdilution test was conducted according to the CLSI M7-A9 and M27-A3 reference methods. After screening, microbial species with minimum inhibitory concentration (MIC) values were selected for biofilm tests. These tests evaluated biomass using the crystal violet (CV) test, metabolic activity using the MTT assay, and structural analysis via Scanning Electron Microscopy (SEM). Cytotoxicity was evaluated in human gingival fibroblasts (FMM-1). There were reductions of 29.4 and 42.7% in CV and MTT assays for Candida spp. biofilm. S. mutans and P. aeruginosa biofilms showed a decrease of 15.7 and 28.6%, respectively. Cell viability tests indicated 55.1, 56.9, and 65.5% of viability after contact with 1.93, 0.96, and 0.48 mg/mL of the extract, respectively. The P. paniculata extract showed antimicrobial action, displayed MIC values, and antibiofilm action on P. aeruginosa, S. mutans, and C. albicans. The cytotoxicity on the FMM-1 cell line was dose-dependent. Therefore, P. paniculata extract holds significant potential for developing new drugs.