Balamuthia mandrillaris is the causative agent of granulomatous amoebic encephalitis, a rare and often fatal infection affecting the central nervous system. The amoeba is isolated from diverse environmental sources and can cause severe infections in both immunocompromised and immunocompetent individuals. Given the limited understanding of B. mandrillaris, our research aimed to explore its protein profile, identifying potential immunogens crucial for early granulomatous amoebic encephalitis diagnosis. Cultures of B. mandrillaris and other amoebas were grown under axenic conditions, and total amoebic extracts were obtained. Proteomic analyses, including two-dimensional electrophoresis and mass spectrometry, were performed. A 50-kDa band showed a robust recognition of antibodies from immunized BALB/c mice; peptides contained in this band were matched with elongation factor-1 alpha, which emerged as a putative key immunogen. Besides, lectin blotting revealed the presence of glycoproteins in B. mandrillaris, and confocal microscopy demonstrated the focal distribution of the 50-kDa band throughout trophozoites. Cumulatively, these observations suggest the participation of the 50-kDa band in adhesion and recognition mechanisms. Thus, these collective findings demonstrate some protein characteristics of B. mandrillaris, opening avenues for understanding its pathogenicity and developing diagnostic and therapeutic strategies.