Background: Orthopedic surgeons often use the intra-articular white blood counts (WBCs) and the percentage of polymorphonuclear cells (PMN) in the diagnosis of an acute swollen and painful knee joint in children. Today, there is no established threshold for the synovial WBC, and their differentiation, as indicative of native joint knee bacterial arthritis. We determine the sensitivity and specificity of synovial WBCs and PMN percentages in the prediction of a community-acquired, acute bacterial native joint septic arthritis (SA) in the pediatric population. Methods: A retrospective study on healthy children 0–16 years of age who underwent knee joint aspiration for a community-acquired, acute irritable knee effusion in our tertiary-care children’s hospital between May 2009 and April 2019 was conducted. We divided the study population into two groups according to the detection of bacterial arthritis in the synovial fluid (bacterial arthritis versus its absence) and compared the intra-articular leukocyte and C-reactive protein (CRP) levels. Results: Overall, we found a statistically significant difference regarding the total CRP (p = 0.017), leukocyte or PMN counts (p ≤ 0.001 in favor of a bacterial arthritis). In contrast, the percentage of the neutrophils was not determinant for the later confirmation of bacterial pathogens, and we were unable to establish diagnostically determining minimal thresholds of the intra-articular CRP and leukocyte levels. Conclusions: This pilot study suggests that either the leukocyte or PMN counts may be associated with a bacterial origin of knee arthritis in children. We plan a larger prospective interventional study in the future to confirm these findings including the investigation of other joint aspirate biomarkers.