Pharmaceutical residues and their transformation products (TPs) have been found in wastewaters and aquatic environments, and they cause adverse effects in aquatic organisms, especially upon chronic exposure and even at very low doses. Cyclophosphamide (CP) is a common alkylating agent that is effective against a wide variety of tumors. L-ascorbic acid is a known water-soluble vitamin that has antioxidant properties. The pharmaceutical residues of both drugs are detected in the environment, and as biologically active molecules they have the ability to affect organisms. In this work we presented the use of a microbiological biosensor E. coli K-12/recA-gfpmut2 in the assessment of the effects of residues of cyclophosphamide and vitamin C and their mixtures on living cells. In our studies, we have shown that mixtures of CP and vitamin C in the highest CP concentrations are more toxic to E. coli strain compared to parent chemicals. In addition, we also found that drug mixtures, especially at the highest CP concentrations, more strongly stimulate the recA promoter and gfp gene expression. We showed that an Escherichia coli K-12/recA-gfpmut2 microbial biosensor is a useful tool in assessing the impact of pharmaceutical residues of cyclophosphamide and L-ascorbic acid and their mixture on living bacteria cells. A stronger impact and toxicity of the mixtures of both tested chemical compounds was found in comparison to parent chemicals.