Green synthesized silver nanoparticles (AgNPs) have been used against antibiotic-resistant bacteria and chemo-resistant cancer cells. We synthesized AgNPs from Acacia nilotica pods, evaluating their antibacterial activity against eight bacterial strains and anticancer efficiency against two colon cancer cell lines, SW620 and SW480. Expression levels of eight genes (β-catenin, APC, TP53, Beclin1, DKK3, Axin, Cyclin D1, and C-myc) were checked by a reverse transcription-polymerase chain reaction in cancer cells before and after treatment with A. nilotica extract and A. nilotica-AgNPs. Prepared nanoparticles were characterized through ultraviolet-visible (UV-vis), Zetasizer, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Fourier transform infrared spectroscopy (FTIR) was used to identify the functional group in extracts. At first, AgNPs were confirmed by a sharp peak of surface plasmon resonance at 375 nm. The Z-average size was 105.4 nm with a polydispersity index of 0.297. TEM showed particle size of 11–30 nm. The prepared AgNPs showed promising antibacterial activity against bacterial strains and cytotoxic activity against the cancer cell lines. Expression levels of all the genes were affected by extract and AgNPs treatment. Overall, this study recommended both A. nilotica pods and A. nilotica-AgNPs as attractive candidates for antibacterial and anticancer applications.