Nanotechnology holds significant promise for the development of novel and necessary products that enhance human health. Pharmacology and nanotechnology have contributed to developing advanced and highly effective drugs for cancer treatment and combating microbial infections. The microbiological effectiveness against the variety of examined microorganisms was assessed using the time killer curve, scanning electron microscopy (SEM), MIC techniques, and the agar well diffusion method. SEM was utilized to enhance the analysis of the mechanisms underlying the bio-interface interaction and intracellular localization of calcium oxide nanoparticles (CaONPs). The MTT test was used to examine the cytotoxicity of CaONP anticancer activity in various cancer cells, including colon, breast, and hepatic cells. The efficacy of CaONPs as an anticancer medication was elucidated by analyzing the gene expression of both treated and untreated cancer cells. MIC and MBC of CaONPs against Escherichia coli and Staphylococcus epidermidis were 150, 150, 150, and 200 µg/ml, respectively. The MIC and MFC of CaONPs against Candida albicans were 200 µg/ml and 250 µg/ml, respectively. The IC50 values of various CaONPs vary depending on the type of cancer cells. The gene expression analysis of breast cancer cells undergoing treatment revealed the identification of several cancer-controlling genes, namely BAX, BCL2, P53, TERT, KRAS1, KRAS2, and RB1. The study demonstrated the notable antibacterial efficacy of CaONPs, highlighting their potential as cancer therapies.