In the present research work, copper oxide-titanium dioxide nanocomposites were synthesized for the first time using advanced pulsed laser ablation in liquid (PLAL) technique for disinfection of drug-resistant pathogenic waterborne biofilm-producing bacterial strains. For this, a series of copper oxide-titanium dioxide nanocomposites were synthesized by varying the composition of copper oxide (5%, 10%, and 20%) with titanium dioxide. The pure titanium dioxide and copper oxide-titanium dioxide nanocomposites were characterized by advanced instrumental techniques. XRD, TEM, FE-SEM, EDX, elemental mapping and XPS analysis results consistently revealed the successful formation of copper oxide-titanium dioxide nanocomposites using PLAL technique. The antibacterial and antibiofilm activities of pure titanium dioxide and copper oxide-titanium dioxide nanocomposites were investigated against biofilm-producing strains of Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa by various methods. Our results revealed that the PLAL synthesized copper oxide-titanium dioxide nanocomposites showed enhanced anti-biofilm and antibacterial activity compared to pure titanium dioxide in a dose-dependent manner against targeted pathogens. Furthermore, the effects of pure titanium dioxide and copper oxide-titanium dioxide nanocomposites on bacterial morphology, biofilm formation, aggregation and their colonization by targeted pathogens were also examined using scanning electron microscopy. Microscopic images clearly showed that the cell envelope of almost all the cells were rumples, rough, had irregularities and abnormal appearance with the major damage being characterized by the formation of "pits". Many depressions and indentations were also seen in their cell envelope and the original shape of Pseudomonas aeruginosa cells changed from normal rod to swollen, large and elongated which indicates the loss of membrane integrity and damage of cell wall and membrane. The findings suggested that PLAL synthesized copper oxide-titanium dioxide nanocomposites have good potential for removal of biofilm or killing of pathogenic bacteria in water distribution network and for wastewater treatment, hospital and environmental applications. In addition, cytotoxic activity of pure TiO and PLAL synthesized copper oxide-titanium dioxide nanocomposites against normal and healthy cells (HEK-293) and cancerous cells (HCT-116) were also evaluated by MTT assay. The MTT assay results showed no cytotoxic effects on HEK-293 cells, which suggest TiO and PLAL synthesized copper oxide-titanium dioxide nanocomposites are non-toxic to the normal cells.