Septic arthritis is a medical emergency requiring early microbiological diagnosis and immediate initiation of appropriate antibiotic therapy to prevent irreversible joint damage. This 9-year retrospective study aimed to describe the epidemiological and microbiological features of culture-confirmed native joint septic arthritis cases diagnosed at the bacteriology laboratory of the Mohammed V Military Instruction Hospital in Rabat (2016–2024), and to evaluate antimicrobial susceptibility profiles of isolated bacteria. Of 922 synovial fluid samples analyzed, 64 culture-confirmed septic arthritis cases were retained. Cytobacteriological evaluation included macroscopic examination, leukocyte count, microcrystal search, Gram staining, and culture on solid enriched media complemented by liquid enrichment broth (brain-heart infusion) and/or inoculation into blood culture bottles. Antimicrobial susceptibility was assessed using the disk-diffusion method following the guidelines of the European Committee on Antimicrobial Susceptibility Testing. The median patient age was 62 years [IQR 44–72], with a male predominance (male-to-female ratio of 2.2). The overall culture positivity rate was 6.9% (64/922). Gram staining was positive in 63% of cases (40/64), predominantly revealing Gram-positive cocci (59%, 38/64). Primary culture was positive in 86% of cases (55/64), while enrichment techniques enabled pathogen isolation in 9 initially sterile samples. Gram-positive cocci accounted for 85% of isolates (57/67), led by Staphylococcus aureus (43%, 29/67), followed by streptococci (27%, 18/67) and Gram-negative bacilli (12%, 8/67). Four multidrug-resistant strains were identified: Three methicillin-resistant Staphylococcus aureus (MRSA, 10% of S. aureus, 3/29) and one extended-spectrum beta-lactamase producing Klebsiella pneumoniae. Native joint septic arthritis in our setting is primarily caused by Staphylococcus aureus, with low multidrug-resistant organism prevalence. A rigorous cytobacteriological approach (including systematic enrichment techniques) is essential to optimize diagnostic yield. These findings support empirical antibiotic therapy primarily targeting Gram-positive cocci, guided by local ecology, with regular protocol reassessment in line with evolving resistance patterns.