A series of O-phenanthroline silver(I) complexes were synthesized and characterized by infrared (IR) spectroscopy, mass spectrometry (MS), 1H nuclear magnetic resonance (NMR) spectroscopy and single-crystal X-ray crystallography. The cytotoxicity of the silver(I) complex (P-131) was evaluated in the cancer cell lines HCT-116, HeLa, and MDA-MB-231 and the normal cell line LO2 via MTT assays. The 50% inhibition concentration (IC50) of P-131 on HCT116 cell line is 0.86 & PLUSMN; 0.03 & mu;M. It is far lower than the IC50 value of cisplatin (9.08 & PLUSMN; 1.10 & mu;M), the IC50 value of normal cell LO2 (76.20 & PLUSMN; 0.48 & mu;M) is much higher than that of cisplatin (3.99 & PLUSMN; 0.74 & mu;M), indicating that its anticancer effect is stronger than that of cisplatin, and its biological safety is greater than that of cisplatin. Furthermore, anticancer mechanistic studies showed that P-131 inhibited cell proliferation by blocking DNA synthesis and acted temporally on the nucleus in dividing HCT-116 cells. Moreover, P-131 increased intracellular reactive oxygen species (ROS) levels in a dose-dependent manner. Notably, 10 mg/kg P131 showed better antitumor effects than oxaliplatin in an HCT116 human colorectal xenograft mouse model without inducing toxicity. Moreover, the microdilution broth method was used to evaluate the antimicrobial properties of P-131 against Pseudomonas aeruginosa and Candida albicans. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy.