This study evaluated in vitro antibacterial, antifungal, anticancer and antioxidant activities of methanolic leaf and root extracts from Solanum nigrum L. and determined its chemical composition. The chemical profile of S. nigrum L. extract was tested using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Disc diffusion and microdilution assays were used for the antibacterial activities. Antifungal activity was measured using the poisoned food technique. In vitro activity on the cell culture model was assessed by MTT assay, viability measurement and by determination of cellular ROS by DCFDA assay. The minimal inhibitory concentrations for extracts from aerial parts ranged from 125 to 500 μg/mL for gram-positive cocci and Pectobacterium strains. The growth inhibition coefficient ranged from 17–56%, depending on the dosage. The antifungal effect of S. nigrum extracts on the tested filamentous fungi depended on the dose. An inhibitory effect of 50–56% on fungi was observed against Alternaria alternata and Chaetomium globosum. The study showed a reduction in cellular vitality of THP1, A549, MCF7 and HeLa cancer lines using both extracts. In addition, there was a decrease in the number of viable cells in cultures incubated with the extract of aerial parts and a reduction in oxygen radicals in the cells. The obtained results indicate the possibility of using S. nigrum extracts from the aerial part as antimicrobial factors. Both extracts show chemopreventive properties by inhibiting the growth of cancer cells and reducing the level of free radicals. Both extracts show chemopreventive properties by inhibiting cancer cell growth and lowering free radical levels. The broad biological activity of the studied extracts can be used in agriculture, veterinary usages and medicine.