Orthopedic and dental implants made from Ti6Al4V are widely used due to their excellent mechanical properties and biocompatibility. However, the long-term performance of these implants can be compromised by bacterial infections. This study explores the development of hierarchically textured surfaces with enhanced bactericidal properties to address such challenges. Hierarchical surface structures were developed by combining microscale features produced by a microsecond laser and superimposed submicron features produced using a femtosecond laser. Microscale patterns were produced by the pulsed laser surface melting process, whereas submicrometer laser-induced periodic surface structures were created on top of them by femtosecond laser processing. Escherichia coli bacterial cells were cultured on the textured surface. After 24 h, a staining analysis was performed using SYTO9 and PI dyes to investigate the samples with a confocal microscope for live dead assays. Results showed bacterial colony formation onto the microscale surface textures with live bacterial cells, whereas the hierarchical surface textures display segregated and physically damaged bacterial cell attachments on surfaces. The hierarchical surface textures showed ∼98% dead bacterial cells due to the combined effect of its multiscale surface features and oxide formation during the laser processing steps. The efficacy of hierarchical surface textures in enhancing the antibacterial behavior of Ti6Al4V implants is evident from the conducted research. Such laser-based surface treatments can find potential applications in different industrial sectors.