Aluminum-doped zinc oxide (ZnO:Al) thin films were deposited onto glass substrates using spray pyrolysis technique with the substrate temperature of 400 °C. X-ray diffraction analysis indicated that the films were polycrystalline with hexagonal wurtzite structure preferentially oriented along (002) direction. Surface morphology of the films obtained by scanning electron microscopy showed that the grains were of nanoscale size with porous nature for 6 at.% of Al. Atomic force microscopy observations revealed that the particles size and surface roughness of the films decreased with Al-doping. Optical measurements indicated that ZnO:Al (6 at.%) exhibited a band gap of 3.11 eV, which is lower than that of pure ZnO film, i.e. 3.42 eV. Photoluminescence analysis showed weak NBE emission at 396 nm for Al-doped films. The low resistivity, high hall mobility and carrier concentration values were obtained at a doping ratio of 6 at.% of Al. The effective incorporation of 6 at.% of Al into ZnO lattice by occupying Zn sites yielded a well-pronounced antibacterial activity against Staphylococcus aureus.