Microbiological diagnosis of osteoarticular infections (OI) is crucial for a successful treatment. A prospective multicenter study including 262 synovial fluids with suspicion of acute OI was performed between July 2021 and October of 2022. BioFire Joint Infection Panel multiplex-PCR test was performed and results were compared with conventional cultures of synovial fluid specimens. In total, 136 microorganisms were detected, and fourteen samples were positive for more than one microorganism. In monomicrobial infections (n = 87) agreement with culture was 69%. In 26 samples, the multiplex PCR yield an additional positive result when culture result was negative. It helped in the detection of fastidious microorganisms as K. kingae and N. gonorrhoeae. This multiplex PCR has proven to be a useful technique that can be used for patients with high suspicion of acute OI in a rapid and automated manner.