###### Abstract  
Bimetallic nanoparticles (BMNPs) combine unique and synergistic properties of two metals, allowing new specific applications. In this study, bimetallic AuFe nanoparticles and their conjugates with methotrexate (MTX) were obtained with an environmentally safe method of metal-vapor synthesis. The composition and electronic structure of the particles were investigated with X-ray photoelectron spectroscopy (XPS), energy dispersive X-ray (EDX) spectrum and X-ray absorption spectroscopy (XANES and EXAFS). The effects of BMNP-MTX conjugates on human primary cells and tumor cell lines were evaluated with neutral red uptake and MTT in vitro cytotoxicity assays. Bright-field microscopy analyses of tumor spheroid size and evaluations of tumor spheroid vitality based on SFDA AM staining were carried out. In vitro assays for an antibacterial activity evaluation of the generated samples were performed. The influence of BMNP-MTX on cytokine production with normal leukocytes was assessed using ELISA. X-ray analyses of the samples demonstrated that gold was in the ground state Au\({}^{0}\) as well as Au\({}^{+}\) and Au\({}^{3+}\) states are present in small quantities, whereas iron existed as a mixture of non-histometric oxides with states close to Fe\({}^{2+}\) and Fe\({}^{3+}\). The modification of the AuFe system with MTX is accompanied by a threefold increase in the relative proportion of the Au\({}^{+}\) state. BMNP-MTX conjugates demonstrated significant antitumor activity compared to the drug alone, which proves the ability of the generated nanoconjugates to improve the effectiveness of MTX therapy. This was confirmed by a marked reduction in the size and vitality of AuFe-MTX-treated 3D tumor spheroids. In addition to their selective antitumor activity, AuFe-MTX exhibited moderate antibacterial activity and induced sample-specific cytokine production with hormonal human leukocytes--which points to an immunostimulatory potential. The present findings indicate important and diverse biological properties of BMNP-MTX conjugates and thus highlight perspectives for their biomedical applications and new immune-specific abilities. bimetallic nanoparticles; gold; iron oxide; methotrexate; antitumor activity; antibacterial properties; immunostimulatory potential 2023 characteristics and abilities for diverse functionalization makes them promising tools for the creation of new drugs, improvement of standard drug's effectiveness and application in clinical practice [7]. They could serve as convenient drug carriers and, in addition, could exhibit therapeutic potential depending on their specific composition and properties and thus enhance current therapeutic strategies and contribute to the development of new ones. A number of studies have already demonstrated the ability of BMNPs to serve as drug delivery systems, photothermal ablation and magnetic hyperthermia agents [5]. They also serve as versatile tools for diagnostical bioimaging [6], theranostics [5], immunotherapy [8] and the generation of improved and more sophisticated biosensors [6]. Bimetallic nanosystems containing iron are among the most studied objects whose properties can be regulated by a modification with other metals [9]. Heterometallic nanoparticles containing gold and iron demonstrate improved optical and magnetic properties [10] and hence improve their efficient application in many fields [11], including medicine [5; 12]. The introduction of gold into iron oxide nanoparticles does not reduce the magnetic characteristics of the system and allows the use of external magnetic influence to control the AuFe system as a whole [5; 13]. Remarkable magnetic properties have been demonstrated for AuFe alloy nanoparticles, which makes them promising candidates for application as contrast agents in computed tomography (CT) and magnetic resonance imaging (MRI) diagnostic techniques [13]. Recent studies have proven the benefits of AuFe BMNPs and their potential use in magnetic guidance and magnetic hyperthermia therapy [14; 15].