The important role of miRNA in cell proliferation and differentiation has raised interest in exogenous ribonucleases (RNases) as tools to control tumour‐associated intracellular and extracellular miRNAs. In this work, we evaluated the effects of the RNase binase from Bacillus pumilus on small non‐coding regulatory RNAs in the context of mouse RLS40 lymphosarcoma inhibition. In vitro binase exhibited cytotoxicity towards RLS40 cells via apoptosis induction through caspase‐3/caspase‐7 activation and decreased the levels of miR‐21a, let‐7g, miR‐31 and miR‐155. Intraperitoneal injections of binase in RLS40‐bearing mice resulted in the retardation of primary tumour growth by up to 60% and inhibition of metastasis in the liver by up to 86%, with a decrease in reactive inflammatory infiltration and mitosis in tumour tissue. In the blood serum of binase-treated mice, decreases in the levels of most studied miRNAs were observed, excluding let‐7g, while in tumour tissue, the levels of oncomirs miR‐21, miR‐10b, miR‐31 and miR‐155, and the oncosuppressor let‐7g, were upregulated. Analysis of binase‐susceptible miRNAs and their regulatory networks showed that the main modulated events were transcription and translation control, the cell cycle, cell proliferation, adhesion and invasion, apoptosis and autophagy, as well as some other tumour‐related cascades, with an impact on the observed antitumour effects. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.