A bstract Background Isolation of the causal microorganisms in osteomyelitis presents a major challenge for treating clinicians. Several methods have been proposed to rapidly and accurately identify microorganisms. There has been an increasing interest in using Raman spectroscopy in the field of microbial detection and characterisation. This paper explores the use of Raman spectroscopy identification as one of the most difficult-to-isolate microorganisms causing osteomyelitis. Methods and results Fresh healthy human bone samples were collected from patients undergoing a total knee replacement. These samples were then inoculated with fresh overnight Pseudomonas aeruginosa (PAO) cultures. Bacteria growth and bone ultrastructural changes were monitored over a period of 6 weeks. The experiment demonstrated ultrastructural bony destruction caused by osteolytic PAO secretions. Raman-specific spectral signatures related to the cellular membranes of PAO structures were spotted indicating survival of bacteria on the bone surface. Conclusion This study showed the promising ability of Raman spectroscopy to identify the presence of bacteria on the surface of inoculated bone samples over time. It was able to detect the osteolytic activity of the bacteria as well as ultrastructure specific to PAO virulence. This method may have a role as an aid to existing diagnostic methods for fast and accurate bacterial identification in bone infection. How to cite this article Al Ghaithi A, Al Bimani A, Al Maskari S. Investigating the Growth of Pseudomonas aeruginosa and Its Influence on Osteolysis in Human Bone: An In Vitro Study. Strategies Trauma Limb Reconstr 2021;16(3):127–131.