Cryptococcus neoformans and Cryptococcus gattii are the leading causative agents of cryptococcosis, an invasive and potentially fatal infectious disease. Therapeutic failures are due to the increase in antifungal resistance, the adverse effects of drugs and the unavailability of therapeutic regimens in low-income countries, which limits the treatment of cryptococcosis, increasing the morbidity and mortality associated with these infections. Thus, new antifungal drugs and innovative strategies for the cryptococcosis treatment are urgently needed. The aim of the present study was to evaluate the effect of synthetic thiourea RTB 168 on planktonic C. neoformans and C. gattii. Furthermore, the interaction between the RTB 168 and amphotericin B (AmB) was evaluated in vitro. The effect of compounds combination was evaluated by the two-dimensional checkerboard microdilution method. The nature of interactions of the compounds was analyzed by the fractional inhibitory concentration (FIC) index (FICI) which is defined as the sum of FICs values of both compounds. FIC was defined as the minimum inhibitory concentration (MIC) of each compound in combination divided by the MIC of the compound used alone. The combination between RTB 168 and amphotericin B exhibited a synergistic fungicidal activity with FICI value equal to 0.08 for C. neoformans and C. gattii strains except for C. gattii 62752 (FICI = 0.15). The fungicidal effect for this treatment was supported by the analysis of cell viability using fluorescent dyes. The images show that untreated yeasts exhibited red fluorescent structures in their cytoplasm, indicating metabolically active cells with intact cytoplasmic membranes. However, cells treated with RTB 168, AmB and the combination MIC exhibited diffuse green staining indicating cell death. To observe the effect of the treatments on Cryptococcus spp. capsule, the thickness of this structure was measured by using light microscopy in yeast cells negatively stained with Chinese ink. Untreated cells were typically round and were surrounding by a clear capsule. The treatments with RTB 168 and AmB, alone or in combination, lead to a significant (p < 0.05) reduction in capsule and cell size after 48 h of incubation when compared to the untreated control. These findings indicate that RTB 168 may be an important source of new compounds which can be used as a fungal-specific adjuvant for the treatment of cryptococcosis. The combination may be one of the feasible ways to overcome antimicrobial resistance by reducing drug concentration and side effects.