Background: Ischemic stroke is one of the leading causes of mortality and disability. The neuroimaging methods are the gold standard for diagnostics. Biomarkers of cerebral ischemia are considered to be potentially helpful in the determination of the etiology and prognosis of patients with ischemic stroke. Aim: This study aimed to investigate the usefulness of serum S100B protein levels as a short- and long-term prognostic factor in patients with ischemic stroke. Study design and methods: The study group comprised 65 patients with ischemic stroke. S100B protein levels were measured by immunoenzymatic assay. Short-term functional outcome was determined by the NIHSS score on day 1 and the difference in the NIHSS scores between day 1 and day 9 (delta NIHSS). Long-term outcome was assessed by the modified Rankin Scale (MRS) at 3 months after the stroke. At the end of the study, patients were divided into groups based on the NIHSS score on day 9 (0–8 “good” and >8 “poor”), the delta NIHSS (“no improvement” ≤0 and >0 “improvement”), and the MRS (“good” 0–2 and >2 “poor”). Differences in S100B levels between groups were analyzed with the ROC curve to establish the optimal cut-off point for S100B. The odds ratio was calculated to determine the strength of association. Correlations between S100B levels at three time points and these variables were evaluated. Results: We revealed a statistically significant correlation between S100B levels at each measurement point (<24 h, 24–48 H, 48–72 h) and the NIHSS score on day 9 (R Spearman 0.534, 0.631, and 0.517, respectively) and the MRS score after 3 months (R Spearman 0.620, 0.657, and 0.617, respectively). No statistically significant correlation was found between S100B levels and the delta NIHSS. Analysis of the ROC curve confirmed a high sensitivity and specificity for S100B. The calculated AUC for the NIHSS on day 9 were 90.2%, 95.0%, and 82.2%, respectively, and for the MRS, 83.5%, 83.4%, and 84.0%, respectively. After determining the S100B cut-off, the odds ratio for beneficial effect (NIHSS ≤ 8 at day 9 or MRS 0–2 after 3 months) was determined for each sampling point. Conclusion: S100B is a useful marker for predicting short- and long-term functional outcomes in patients with ischemic stroke.