Bone and joint infections (BJI) contribute to significant morbidity and mortality. The epidemiology has undergone several changes over last decade, following the advent of molecular diagnostics and non-culture-based methods. A plethora of commensal and fastidious organisms have been implicated in BJIs, which vary in native and prosthetic joints. Common etiological agents include Staphylococcus aureus, Other Staphylococcus sp., Enterococcus sp., Enterobacterales, intracellular bacteria, Mycobacteria, anaerobes, Salmonella, K. kingae and rarely fungi and parasites. Polymicrobial infections are common subsequent to trauma and chronic ulcers, in diabetic patients, while Cutibacterium acnes and Other Staphylococcus sp. are incriminated in prosthetic joint infections. Multiple sampling is suggested for septic arthritis, osteomyelitis and prosthetic joint infections, wherein at least five samples should be collected. Samples obtained from deeper tissues should be preferred, whenever possible. All the sampling should be performed under aseptic conditions, and sample is transported to laboratory within 2 h of collection. Microbiological diagnosis includes direct microscopy using Gram staining, Giemsa staining, potassium-hydroxide calcofluor stain and Ziehl-Neelsen staining, culture, serology, biomarkers and molecular diagnosis. Culture is usually performed on routine blood and MacConkey agar, however, chocolate agar supplemented with PolyViteX and Schaedler agar, RCM, brain heart infusion broth, Schaedler or Rosenow broth should also be inoculated to identify fastidious organisms. Automated blood culture bottles with enhanced diagnostic yield are significant in identification of common and fastidious, slow-growing organisms. Caution is required for extended incubation of bacterial and fungal cultures. Non-culture-based diagnosis with lesser turnaround time (TAT) integrates conventional biomarkers like interferon-gamma release assays (IGRA), IL-6, C-reactive protein, and newer biomarkers like leucocyte esterase, alpha defensin, calprotectin, presepsin and neopterin. Molecular modalities viz. conventional and real-time polymerase chain reaction (PCR), with or without multiplexing, PCR electrospray ionization mass spectrometry have less TAT and better accuracy. The evolving next-generation sequencing techniques like ion-torrent, Illumina, Applied Biosystems Sequencing by oligonucleotide ligation and detection system (SOLiD), oxford nanopore; mNGS and syndromic platforms are being increasingly used for precise identification, with less TAT. Timely precise diagnosis and management of BJIs mandates comprehensive understanding of host-microbe interactions, biofilm dynamics, pharmacokinetics-pharmacodynamics and susceptibility profile of etiological agents.