Topotecan is a cytostatic drug from the camptothecin group, it acts by inhibiting topoisomerase 1 (TOP1). Tyrosyl-DNA phosphodiesterase 1 (TDP1) is capable of interfering with the action of TOP1 inhibitors, reducing their therapeutic efficacy. Suppression of TDP1 activity may enhance the effects of topotecan. In this work, we investigated the effect of the antitumor drug topotecan alone and in combination with a TDP1 inhibitor, a hydrazinothiazole derivative of usnic acid, on Krebs-2 mouse ascites tumors. We have previously shown that this derivative efficiently inhibits TDP1. In the present work, we show that both topotecan and the TDP1 inhibitor have an antitumor effect when evaluated separately. The combination of topotecan and the TDP1 inhibitor additively reduces both the weight of the ascites tumor and the number of cells in ascites. In mice, the TDP1 inhibitor alone or in combination with topotecan eliminated the tumor cells. After the combined intraperitoneal administration of these two compounds, we observed cells in which lipid droplets occupied almost the entire cytoplasm and the accumulation of cell detritus, which was absent in the samples collected from mice treated with each compound separately.