Silver nanoparticle (AgNP) based approaches using plant materials have been accepted as biomedical applications. The current study aimed to test the antibacterial, antibiofilm, and anticancer activity of silver nanoparticles synthesized by seed extract of Nigella sativa (Ns) as stabilizing and reducing agents. Characterization was done through UV−visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electronic microscopy (SEM), and transmission electronic microscopy (TEM) analyses. UV-Vis spectroscopy showed a specific silver plasmon peak at 400 nm and a quick color change was observed in the bio-reaction medium. Electron microscopic images of Ns-AgNPs identified as spherical in shape with varied size ranged between 8 and 80 nm and zeta potential analysis evidenced the particles stability and polydisperity. Antibiofilm activity of Ns-AgNPs was evident as at 12.5 µg/mL Ns-AgNps restricted the biofilm formation by 88.42% for Enterococcus faecalis, 84.92% for E. coli, 81.86% for Klebsiella pneumonia, 82.84% for Staphylococcus aureus, and 49.9% for Pseudomonas aeruginosa, respectively. Furthermore, biologically synthesized AgNPs showed the significant bacteriostatic and bactericidal activity. Even the lowest concentration of Ns-AgNps restricted the highest rate of inhibition against S. aureus (6.5 and 15 µg/mL) and E. faecalis (6.5 and 15 µg/mL). Antimicrobial activity of S. aureus and E. fecalis was more prominent than E. coli (15 and 30 µg/mL), K. pneumonia (15 and 30 µg/mL) and P. aeruginosa (30 and 60 µg/mL) respectively. Moreover, Ns-AgNPs revealed significant cytotoxic ability and substantially killed human breast cancer cell (HCC-712) viability. The results of current study advocate that Ns-AgNps may be considered as a potential option in biomedical applications, alternative therapy, designing anti-biofilm agents, treating multi drug resistance bacterial infection, and anti-cancer therapy.