Escherichia coli and Staphylococcus aureus bacterial retention on mirror-polished and ultrashort pulse laser-textured surfaces is quantified with a new approach based on ISO standards for measurement of antibacterial performance. It is shown that both wettability and surface morphology influence antibacterial behavior, with neither superhydrophobicity nor low surface roughness alone sufficient for reducing initial retention of either tested cell type. Surface structures comprising spikes, laser-induced periodic surface structures (LIPSS) and nano-pillars are produced with 1030 nm wavelength 350 fs laser pulses of energy 19.1 μJ, 1.01 μJ and 1.46 μJ, respectively. SEM analysis, optical profilometry, shear force microscopy and wettability analysis reveal surface structures with peak separations of 20–40 μm, 0.5–0.9 μm and 0.8–1.3 μm, average areal surface roughness of 8.6 μm, 90 nm and 60 nm and static water contact angles of 160°, 119° and 140°, respectively. E. coli retention is highest for mirror-polished specimens and spikes whose characteristic dimensions are much larger than the cell size. S. aureus retention is instead found to be inhibited under the same conditions due to low surface roughness for mirror-polished samples (S_a: 30 nm) and low wettability for spikes. LIPSS and nano-pillars are found to reduce E. coli retention by 99.8% and 99.2%, respectively, and S. aureus retention by 84.7% and 79.9% in terms of viable colony forming units after two hours of immersion in bacterial broth due to both low wettability and fine surface features that limit the number of available attachment points. The ability to tailor both wettability and surface morphology via ultrashort pulsed laser processing confirms this approach as an important tool for producing the next generation of antibacterial surfaces.