Fengjun Xia,1,2,* Xiaoyan Tao,2,3,* Haichen Wang,1,2 Jian Shui,4 Changhang Min,1,2 Yubing Xia,1,2 Jun Li,1,2 Mengli Tang,1,2 ZhaoJun Liu,1,2 Yongmei Hu,1,2 Huidan Luo,1,2 Mingxiang Zou1,2 1National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 2Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, 410008, People’s Republic of China; 3Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China; 4Department of Clinical Laboratory, Changsha Central Hospital, Changsha, 410008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Mingxiang Zou, National Clinical Research Center for Geriatric Disorders, Department of Clinical Laboratory, Xiangya Hospital, Central South University, Changsha, Hunan Province, 410008, People’s Republic of China, Tel/Fax +86-7384327440, Email zoumingxiang@csu.edu.cnPurpose: As an under-explored biomaterial, bacterial biofilms have a wide range of applications in the green synthesis of nanomaterials. The biofilm supernatant of Pseudomonas aeruginosa PA75 was used to synthesize novel silver nanoparticles (AgNPs). BF75-AgNPs were found to possess several biological properties.Methods: In this study, we biosynthesized BF75-AgNPs using biofilm supernatant as the reducing agent, stabilizer, and dispersant and investigated their biopotential in terms of antibacterial, antibiofilm, and antitumor activities.Results: The synthesized BF75-AgNPs demonstrated a typical face-centered cubic crystal structure; they were well dispersed; and they were spherical with a size of 13.899 ± 4.036 nm. The average zeta potential of the BF75-AgNPs was − 31.0 ± 8.1 mV. The BF75-AgNPs exhibited strong antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase Escherichia coli (ESBL-EC), extensively drug-resistant Klebsiella pneumoniae (XDR-KP), and carbapenem-resistant Pseudomonas aeruginosa (CR-PA). Moreover, the BF75-AgNPs had a strong bactericidal effect on XDR-KP at 1/2 × MIC, and the expression level of reactive oxygen species (ROS) in bacteria was significantly increased. A synergistic effect was observed when the BF75-AgNPs and colistin were used for the co-treatment of two colistin-resistant XDR-KP strains, with fractional inhibitory concentration index (FICI) values of 0.281 and 0.187, respectively. Furthermore, the BF75-AgNPs demonstrated a strong biofilm inhibition activity and mature biofilm bactericidal activity against XDR-KP. The BF75-AgNPs also exhibited a strong antitumor activity against melanoma cells and low cytotoxicity against normal epidermal cells. In addition, the BF75-AgNPs increased the proportion of apoptotic cells in two melanoma cell lines, and the proportion of late apoptotic cells increased with BF75-AgNP concentration.Conclusion: This study suggests that BF75-AgNPs synthesized from biofilm supernatant have broad prospects for antibacterial, antibiofilm, and antitumor applications.Graphical Abstract: Keywords: multidrug resistant, green synthesis, colistin, metallic silver, melanoma