Antimicrobial resistance (AMR) is projected to result in 10 million deaths every year globally by 2050. Without urgent action, routine orthopaedic operations could become high risk and musculoskeletal infections incurable in a “post-antibiotic era.” However, current methods of studying AMR processes including bacterial biofilm formation are 2D in nature, and therefore unable to recapitulate the 3D processes within in vivo infection. Within this study, 3D printing was applied for the first time alongside a custom-developed bioink to bioprint 3D bacterial biofilm constructs from clinically relevant species including Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA), Escherichia coli and Pseudomonas aeruginosa. Bacterial viability and biofilm formation in bioprinted constructs was excellent, with confocal laser scanning microscopy (CSLM) used to demonstrate biofilm production and maturation over 28 days. Bioprinted 3D MRSA and MSSA biofilm constructs had greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Raman spectroscopy was also adapted in a novel approach to non-invasively diagnose 3D bioprinted biofilm constructs located within a joint replacement model. In conclusion, mature bacterial biofilm constructs were reproducibly 3D bioprinted for the first time using clinically relevant bacteria. This methodology allows the study of antimicrobial biofilm penetration in 3D, and potentially aids future antimicrobial research, replicating joint infection more closely than current 2D culture models. Furthermore, by deploying Raman spectroscopy in a novel fashion, it was possible to diagnose 3D bioprinted biofilm infections within a joint replacement model