© 2021 Hydrogen Energy Publications LLCMetallic nanoparticles (MNPs) have important applications in medicine and technology. Bimetallic NPs, which are among the metallic nanoparticles, are of great interest due to their properties. An innovative method by green synthesis has been developed to obtain bimetallic NPs. Aromatic plants are used in this synthesis method. One of the plants used for green synthesis is Nigella sativa and it has a unique place among plants for use as medicine. In this study, the synthesis of Palladium–Platinum bimetallic nanoparticles (PdPt NPs) and the catalytic, antibacterial, and anticancer activity of synthesized PdPt NPs by green synthesis method using Nigella sativa seed extract are reported. The synthesized PdPt NPs were characterized by Fourier Transform Infrared Spectrophotometer (FTIR), Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), and UV–Vis spectrometry techniques. The catalytic activity of PdPt NPs was determined by performing sodium borohydride (NaBH4) hydrolysis experiments. According to the results obtained, Turnover Frequency (TOF), activation energy, entropy, and enthalpy values were found to be 1664.76 h−1, 13.93 kJ/mol, −119.02 J/mol.K, and 11.43 kJ/mol, respectively. It was determined that PdPt NPs are highly effective catalysts for hydrogen production. PdPt NPs (200 μg/mL) were determined to have antibacterial activity of 57.58%, 64.42%, 48.68%, and 58.77% against Escherichia coli, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, and Bacillus subtilis bacteria, respectively. In addition, the cytotoxic effects of PdPt NPs, MTT against human breast cancer cell line (MDA-MB-231), human endometrial carcinoma cell line (Ishikawa, ISH), human cervical cancer cell line (HeLa), L929-Murine fibroblast cell line test, and IC50 values were calculated. The IC50 values of PdPt NPs applied against MDA-MB-231, ISH, and HeLa cancer cell lines were calculated as 9.1744 ± 1.566 μg/mL, 12.2431 ± 1.132 μg/mL, 18.1963 ± 1.730 μg/mL, respectively. No significant cytotoxic effect was observed in healthy L929-murine fibroblasts. Green synthesis of PdPt NPs was determined to have significant advantages over chemical approaches. The biogenic PdPt NPs synthesized in this study suggest the design of bio-based bimetallic catalysts with high catalytic performance to prevent environmental pollution.