The urinary tract diseases pose an increasing challenge due to the aging of population. We aimed to find new therapeutic approaches, thus we analysed the effect of human amniotic membrane-derived (AM) preparations on cancer urothelial cells and uropathogenic bacteria. Using different cell-biological, molecular and microbiological approaches we analysed the effect of AM scaffolds, AM homogenate and AM-derived cells on T24 cells, derived from human muscle-invasive bladder cancer. We demonstrated that 1) AM scaffolds diminish the proliferation and reduce the invasive potential of T24 cells and contribute to their mesenchymal-to-epithelial transformation, 2) AM homogenate diminishes the proliferation and causes detachment of T24 cells and prevents their attachment to the surface and 3) AM-derived cells diminish the proliferation of T24 cells. The antimicrobial effect of AM preparations was tested on 25 strains of uropathogenic bacterial species. We demonstrated that patches of fresh (fAM) and cryopreserved AM (cAM) have no antimicrobial effect, while the fAM and cAM homogenates have antimicrobial effect on 16 out of 25 tested strains of uropathogenic bacterial species. Moreover, cAM homogenate has antimicrobial effect also on bacteria-infected in vitro models of normal and cancerous urothelium. The manner of preparation and storage of AM affects the range of its antimicrobial effect. Findings that AM preparations inhibit cancer urothelial cells and selected strains of uropathogenic bacteria, importantly contribute to to the knowledge of AM activity and serve as a baseline for studies that will evaluate the use of AM in urology.