P. nigrum L. extracts and the piperine alkaloid have important antimicrobial, anti-inflammatory, and antioxidant properties. Therefore, in this study, we evaluated the antimicrobial activity and cytotoxicity of P. nigrum L. extracts and piperine, a compound isolated from the extracts of P. nigrum L. Extracts obtained via maceration, soxhlet, and purification steps, in addition to isolated piperine, were used in this study. Spectroscopic methods, such as nuclear magnetic resonance, scanning electron microscopy, X-ray diffraction, thermogravimetry, and differential scanning calorimetry, were used to characterize piperine. In the microbiological analyses, the extract obtained via maceration-derived sample showed high efficiency in inhibiting Salmonella spp. (MIC Pseudomonas aeruginosa. Favorable inhibition coefficients were also observed against Staphylococcus aureus and Salmonella spp. (MIC P. aeruginosa. Cytotoxicity evaluation assays in cancer cell lines revealed that piperine exhibited inhibitory potential on all tested tumor cell lines, causing a decrease in cell viability and achieving an IC50 of less than 30 μg/mL. The analyzed extracts from P. nigrum L. seeds showed cytotoxic activity against tumor and non-tumor cell lines.