In the past few decades, titanium and its alloys have been widely used in the orthopaedic field. However, because titanium is bioinert and lacks antibacterial properties, infection may happen when bacteria attach to implant surfaces and form biofilms. It has been studied that some naturally existing micron-scale topographies can reduce bacterial attachment such as cicada wings and gecko skins. The aim of this in vitro study was to find an implant with good biocompatibility and antimicrobial properties by the modification of micron-scale topographies. In this paper, a femtosecond laser was used to provide microtopography coatings on Ti substrates. The surface morphology of Ti substrates was observed by scanning electron microscopy (SEM). XPS was used to fulfil the chemical compositional analysis. The surface wettability was measured by contact angle measurement system. The effect of microtopography coatings with different surface microstructures on bacterial activities and bone marrow mesenchymal stem cells (BMSC) functions was investigated. The results of in vitro study revealed that microtopography coatings restrain the adhesion of Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis), which are common pathogens of orthopaedic implant infections. In addition, microtopography coatings stimulated BMSC adhesion and proliferation. Our studies suggest that a microtopography-coated sample modified by femtosecond laser showed promising antibacterial properties and favourable biocompatibility. The femtosecond laser technique provides an accurate and valid way to produce microtopography coatings with outstanding biocompatibility and antimicrobial properties, and could be widely used to modify the surface of orthopaedic metal implants with great potential.