Several lines of evidence have clearly demonstrated the role of the tumor microenvironment in favoring the drug resistance of melanoma cells, as well as the progression of this cancer type. Since our previous studies proved that the accumulation of prednisolone disodium phosphate (PLP) in melanoma tissue inhibited tumor growth by exerting anti‑angiogenic effects on the most abundant cells of the tumor microenvironment, tumor‑associated macrophages (TAMs), the present study investigated whether PLP could enhance the cytotoxic effects of doxorubicin (DOX) on B16.F10 murine melanoma cells. To assess the antitumor efficacy of the combined therapeutic approach based on PLP and DOX, we used a co‑culture system composed of bone marrow‑derived macrophages (BMDMs) and B16.F10 murine melanoma cells at a cell density ratio that approximates the melanoma microenvironment in vivo, ensuring the polarization of the BMDMs into TAMs. Thus, we assessed the combined therapeutic effects of PLP and DOX on melanoma cell proliferation and apoptosis, as well as on supportive processes for tumor growth, such as oxidative stress as well as the angiogenic and inflammatory capacity of the cell co‑culture. Our data demonstrated that the cytotoxicity of DOX was potentiated mainly via the anti‑angiogenic activity of PLP in the melanoma microenvironment in vitro. Moreover, the amplitude of the cytotoxicity of the combined treatments may be linked to the degree of the suppression of the pro‑angiogenic function of TAMs. Thus, the potent decrease in the expression of the majority of the angiogenic and inflammatory proteins in TAMs following the concomitant administration of PLP and DOX may be associated with their anti‑proliferative, as well as pro‑apoptotic effects on B16.F10 melanoma cells. However, the combination therapy tested did not affect the immunosuppressive phenotype of the TAMs, as the levels of two important markers of the M2‑like phenotype of macrophages (IL‑10 and Arg‑1) were not reduced or even increased following these treatments. On the whole, the findings of this study indicated that PLP improved the therapeutic outcome of DOX in the melanoma microenvironment via the inhibition of the pro‑angiogenic function of TAMs.