Developing antimicrobial surfaces that combat implant-associated infections while promoting host cell response is a key strategy for improving current therapies for orthopaedic injuries. In this paper, we present the application of ultra-short laser irradiation for patterning the surface of a 3D biodegradable synthetic polymer in order to affect the adhesion and proliferation of bone cells and reject bacterial cells. The surfaces of 3D-printed polycaprolactone (PCL) scaffolds were processed with a femtosecond laser (λ = 800 nm; τ = 130 fs) for the production of patterns resembling microchannels or microprotrusions. MG63 osteoblastic cells, as well as S. aureus and E. coli, were cultured on fs-laser-treated samples. Their attachment, proliferation, and metabolic activity were monitored via colorimetric assays and scanning electron microscopy. The microchannels improved the wettability, stimulating the attachment, spreading, and proliferation of osteoblastic cells. The same topography induced cell-pattern orientation and promoted the expression of alkaline phosphatase in cells growing in an osteogenic medium. The microchannels exerted an inhibitory effect on S. aureus as after 48 h cells appeared shrunk and disrupted. In comparison, E. coli formed an abundant biofilm over both the laser-treated and control samples; however, the film was dense and adhesive on the control PCL but unattached over the microchannels.