CuO nanoparticles (NPs) stand out due to their inherent antibacterial activity, cost-effectiveness, and tunable bandgap, rendering them promising for medical, disinfection, environmental, and industrial applications. Our study aimed to synthesize CuO NPs using fish scale (FS) waste and assess the impact of calcination temperature (100°C, 300°C, 500°C, and 700°C) on their structural and antibacterial properties. XRD analysis confirmed the formation of monoclinic structured CuO, corroborated by XPS analysis indicating the Cu2+ oxidation state. Crystallographic parameters such as lattice dimensions, volume of unit cell, density of unit cell, micro-strain, and dislocation density for all four CuO NPs samples were calculated using established equations. The crystallite size was estimated by employing Scherrer equation, Linear Straight-Line Method (LSLM), Williamson-Hall Method (W-H), Halder-Wagner Method (H-W), Monshi-Scherrer Method (M-S), and Size-Strain Plot (SSP) method. Based on the highest value of coefficient of determination (R2), the appropriateness of the measuring techniques was determined, and the crystallite sizes were found to be 10 nm, 11 nm, 24 nm, and 35 nm, respectively. Particle size was estimated based on the FESEM and TEM images, both of which showed an increase in size with calcination temperature. FTIR detected the presence of amide bands, which originated from the FS and EDX analysis provided qualitative and quantitative elemental measurements. The antibacterial activity was investigated against two gram-negative (E. coli and S. typhi) and two gram-positive (B. megaterium and S. aureus) bacterial strains. Increasing calcination temperature resulted in increased particle size, which led to reduced antibacterial activity.